Conformational change within the extracellular domain of B cell receptor in B cell activation upon antigen binding

Zhixun Shen; Sichen Liu; Xinxin Li; Zhengpeng Wan; Youxiang Mao; Chunlai Chen; Wanli Liu

doi:10.7554/eLife.42271

. 2019 Jul 10;8:e42271. doi: 10.7554/eLife.42271

Conformational change within the extracellular domain of B cell receptor in B cell activation upon antigen binding

Zhixun Shen ^1,^†, Sichen Liu ^1,^†, Xinxin Li ^1,^†, Zhengpeng Wan ¹, Youxiang Mao ¹, Chunlai Chen ², Wanli Liu ^1,^✉

Editors: Arup K Chakraborty³, Michael L Dustin⁴

PMCID: PMC6620044 PMID: 31290744

Abstract

B lymphocytes use B cell receptors (BCRs) to recognize antigens. It is still not clear how BCR transduces antigen-specific physical signals upon binding across cell membrane for the conversion to chemical signals, triggering downstream signaling cascades. It is hypothesized that through a series of conformational changes within BCR, antigen engagement in the extracellular domain of BCR is transduced to its intracellular domain. By combining site-specific labeling methodology and FRET-based assay, we monitored conformational changes in the extracellular domains within BCR upon antigen engagement. Conformational changes within heavy chain of membrane-bound immunoglobulin (mIg), as well as conformational changes in the spatial relationship between mIg and Igβ were observed. These conformational changes were correlated with the strength of BCR activation and were distinct in IgM- and IgG-BCR. These findings provide molecular mechanisms to explain the fundamental aspects of BCR activation and a framework to investigate ligand-induced molecular events in immune receptors.

Research organism: Human

Introduction

Recognition of antigen by B cell receptor (BCR) initiates B cell activation, which ultimately lead to the production of protective antibodies against pathogens (Kurosaki et al., 2010). BCR complex comprises a membrane-bound immunoglobulin (mIg) and a noncovalently linked heterodimer composed of Igα and Igβ in 1:1 stoichiometry of mIg: Igα/β (Tolar et al., 2005; Hombach et al., 1990). Antigen engagement induces phosphorylation of immunoreceptor tyrosine-based activation motifs (ITAMs) in Igα/β by the Src family kinase Lyn, resulting in the triggering of signaling cascades (Pierce and Liu, 2010). However, remain unclear is the molecular mechanism through which BCR extracellular antigen binding signal is transmitted across the membrane to the BCR intracellular ITAMs for the purpose of B cell activation. ‘Conformational change model’ has been proposed to explain the initiation of B cell activation, in which it is supposed that through a series of conformational changes within the BCR complex, the interaction of antigen with the extracellular domain of mIg is transduced to the intracellular domain of BCR (Harwood and Batista, 2010).

Although the lack of structural-based evidence significantly limit our understanding of the conformational information of BCR extracellular domains during the transmembrane initiation of BCR activation, several previous studies support this conformational change model by investigating fluorescence resonance energy transfer (FRET) within BCR cytoplasmic domains (Tolar et al., 2005), FRET between plasma membrane and Igα cytoplasmic domain (Lee and Tolar, 2013), FRET between membrane and cytoplasmic domain of mIgG (Chen et al., 2015) upon antigen engagement, respectively. In these studies, binding of antigen led to a conformational change in the BCR cytoplasmic domains from a closed to an open form (Tolar et al., 2005), an increased distance between membrane and Igα but not Igβ (Lee and Tolar, 2013), and dissociation of mIgG cytoplasmic tail from cell membrane (Chen et al., 2015). In addition to these studies focusing on the conformational changes of the cytoplasmic domains of BCR, it is also reported that the Cμ4 portion in mIgM (and Cγ3 portion in mIgG) of the extracellular domain of BCR is both required and sufficient for antigen-binding induced BCR oligomerization and signaling, suggesting antigen engagement triggered the Cμ4 domain of IgM-BCR (or Cγ3 domain of IgG-BCR) into an orientation in which BCRs are accessible for oligomerization (Tolar et al., 2009). Moreover, in our early studies, using a double strand DNA-based tension gauge tether (TGT) experimental system with defined single molecular forces between BCR and surface-immobilized antigen, we observed that IgM- and IgG-BCR exhibited distinct mechanical force sensitivity during activation. IgM-BCR activation was dependent on mechanical force and exhibited a multi-threshold dependence. In contrast, the activation of IgG-BCR only required a low threshold of less than 12 pN (Wan et al., 2015; Wan et al., 2018; Wang and Ha, 2013). Based on the finding that BCR activation was dependent on mechanical forces, it is reasonable to hypothesize that mechanical force delivered by antigen engagement may induce a potential conformational change within BCR complex, which in turn can trigger the transmission of the physical signal outside of the plasma membrane to chemical signal inside of the membrane. Last but not least, antigen binding might induce conformational change of BCR through modulations in the microenvironment (such as cytoskeleton [Mattila et al., 2013] or lipid bilayer [Sohn et al., 2006]) or altering the charge-charge interactions within BCR complex.

All these indicated the importance to address a long standing question in antigen receptor biology: How the extracellular antigen binding signal at the variable region of mIg is transduced to the intracellular ITAMs at the cytoplasmic domain of Igα/β within BCR complex. In detail, whether or not the conformational change occurs within the extracellular domains of BCR upon antigen binding? If yes, where is the exact location of such conformational change; is it at the region within the heavy chain of BCR mIg or between mIg and Igα/β? Whether or not the conformational change within mIg of BCR complex also occurs in soluble Ig molecule upon antigen binding? Whether or not the conformational change of BCR is correlated to the strength of B cell activation? Here, we combined site-specific labeling methodology and FRET-based assay to monitor the conformational changes in the extracellular domains within BCR complex upon antigen binding. Conformational changes within mIg and in the spatial relationship between mIg and Igβ were captured and quantified, and different isotyped BCRs displayed distinct conformational change patterns upon antigen binding. Meanwhile, these antigen-binding induced conformational changes in IgM-BCR on cell membrane were not observed in the case of soluble IgM monomer antibodies. In addition, the correlation between conformational change and the strength of BCR activation was analyzed. All these results may provide molecular explanations for fundamental aspects of BCR activation during transmembrane signaling transduction of B cells. Moreover, experimental systems developed in the present study may also provide a framework from which to examine ligand-induced intramolecular or intermolecular events in BCR and other immune receptors.

Results

Site-specific labeling in mIg heavy chain of BCR complex

To investigate the potential conformational change within extracellular domains of mIg molecule upon BCR engagement with antigen, we constructed various BCR complexes with short tags inserted that permit targeted incorporation of predefined fluorophores by enzymatic labeling (Zhou et al., 2007) or metal chelation (Griffin et al., 1998) #51]. For IgM-BCR, dually tagged VRC01-IgM-BCR (gp120 antigen-specific) (Wang et al., 2018a) was generated to measure the intramolecular FRET between N terminus and Cμ2 domain within mIgM. Specifically, the ybbR tag (Yin et al., 2006; Munro et al., 2014) was introduced into N terminus of IgM heavy chain in VRC01-BCR to enable the site-specific labeling at the Fab domain of Ig heavy chain. Fc portion labeling was achieved through the tetracysteine tag (Hoffmann et al., 2010; Nuber et al., 2016) insertion in Cμ2 domain of VRC01-IgM-BCR, as described for the detailed amino acid position in the Materials and methods section (Figure 1A). Dually tagged VRC01-IgM-BCR can be successfully expressed and labeled in 293T cells (Figure 1B). Similarly, dually tagged VRC01-IgG-BCR containing ybbR tag in N terminus of IgG heavy chain and tetracysteine tag in Cγ2 domain was constructed (Figure 1—figure supplement 1A,B).

Figure 1. — (A) Schematic representation of dually tagged VRC01-IgM-BCR. ybbR tag inserted in N-terminus of IgM heavy chain labeled by CoA 488 was shown as red circle, while tetracysteine tag in Cμ2 region of IgM heavy chain labeled by ReAsH was shown as brown circle. (B) Representative confocal images of WT and dually tagged VRC01-IgM-BCR expressed in 293T cells. ybbR tag and tetracysteine tag were stained by CoA 488 and ReAsH, respectively. Alexa Fluor 647 (AF647) Fab fragment of goat anti-human IgM Fc5μ was used for BCR staining. Scale bar, 5 μm. (C) WT and dually tagged VRC01-IgM-BCR expressing 293T cells binding with V51 monomer antigen. 293T cells without BCR expression were used as a control. (D) Ca²⁺ mobilization analysis of A20II1.6 B cells expressing WT and dually tagged VRC01-IgM-BCR stimulated by V51 trimer antigen.

Figure 1—figure supplement 1. — (A) Schematic representation of dually tagged VRC01-IgM-BCR. ybbR tag inserted in N-terminus of IgM heavy chain labeled by CoA 488 was shown as red circle, while tetracysteine tag in Cμ2 region of IgM heavy chain labeled by ReAsH was shown as brown circle. (B) Representative confocal images of WT and dually tagged VRC01-IgM-BCR expressed in 293T cells. ybbR tag and tetracysteine tag were stained by CoA 488 and ReAsH, respectively. Alexa Fluor 647 (AF647) Fab fragment of goat anti-human IgM Fc5μ was used for BCR staining. Scale bar, 5 μm. (C) WT and dually tagged VRC01-IgM-BCR expressing 293T cells binding with V51 monomer antigen. 293T cells without BCR expression were used as a control. (D) Ca²⁺ mobilization analysis of A20II1.6 B cells expressing WT and dually tagged VRC01-IgM-BCR stimulated by V51 trimer antigen.

Next, we tested whether these modified BCRs expressed in 293T cells or A20II1.6 B cells tolerated the insertion of these short peptide tags. Dually tagged BCRs were expressed in 293T cells for antigen-binding test, while A20II1.6 B cells expressing dually tagged BCRs were used for testing antigen-induced calcium influx. Dually tagged VRC01-IgM-BCR can bind with HIV-1 gp120 antigen (V51 monomer; Wang et al., 2018a) as WT VRC01-IgM-BCR (Figure 1C), and the stimulation by V51 trimer (A foldon trimerization domain of T4 fibritin was fused to the C terminus of V51 for the production of V51 trimer) (Wang et al., 2018a) induced similar calcium influx signal in untagged and dually tagged VRC01-IgM-BCR expressing A20II1.6 B cells (Figure 1D). Similarly, dually tagged VRC01-IgG-BCR expressed in 293T cells or A20II1.6 B cells exhibited comparable function as WT VRC01-IgG-BCR (Figure 1—figure supplement 1C,D). The oligomeric states of V51 trimer and V51 monomer used were determined by blue native-polyacrylamide gel electrophoresis (BN-PAGE) (Figure 1—figure supplement 1E) and confirmed by antigen-induced calcium influx measurement (Figure 1—figure supplement 1F). All these results showed the uncompromised function of dually tagged BCRs to recognize antigen and initiate B cell activation.

Conformational change within the mIgM heavy chain of IgM-BCR upon antigen engagement

Conformational change within mIg heavy chain of antigen-activated VRC01-IgM-BCR was firstly investigated via intramolecular FRET between CoA 488 (CoA-conjugated ATTO 488) label at the N terminus and ReAsH (resorufin arsenical hairpin binder) label in Cμ2 domain (Figure 2A). To exclude the interference of intracellular signaling molecules and prevent competition between exogenous and endogenous BCRs in B cells, FRET experiments were firstly performed in 293T cells. 293T cells expressing dually tagged VRC01-IgM-BCR were stimulated by V51 monomer antigen presented by planar lipid bilayers (Figure 2B). The results showed that the binding of antigen lowered the FRET efficiency between N terminus and Cμ2 domain as measured by the quantification of donor recovery after acceptor bleaching using total internal reflection fluorescence microscopy (TIRFM) (Figure 2C,D). The reduction of FRET signal indicated an increased spatial distance between these two sites upon antigen engagement. To exclude the potential effects from variations in the concentration of donor and acceptor fluorophores, only cells with comparable donor intensity and acceptor intensity were used for analysis in the TIRFM-based acceptor photobleaching FRET experiments (Figure 2D). As a further validation, fluorescence lifetime imaging microscopy FRET (FLIM-FRET) measurement was also performed as it can be applied to determine FRET efficiency irrespectively of the concentration of donor and acceptor fluorophores. Decreased FRET between these two sites within IgM heavy chain resulted from V51 monomer antigen engagement was similarly obtained, as indicated by the increased fluorescence lifetime of CoA 488 (donor chromophore) and thus reduced FLIM-FRET signal after antigen binding (Figure 2E–H). We also repeated the same FRET measurement via acceptor photobleaching method in A20II1.6 B cells to confirm that the reduced FRET signal within the ectodomain of BCR also occurred in B cells (Figure 2I). The increased distance between N terminus and Cμ2 domain of IgM-BCR upon antigen binding suggested that the antigen-induced conformational change might originate at antigen binding site.

Figure 2. — (A) Schematic illustration showing FRET efﬁciency between CoA 488 label at N-terminus (red circle) and ReAsH label at Cμ2 domain (brown circle) of dually tagged VRC01-IgM-BCR. (B) Schematic illustration showing V51 monomer fused with poly-histidine tag presented on the surface of Ni²⁺-containing planar lipid bilayer (PLB) membranes on supported glass coverslips. (**C–D**) Dequenching FRET to measure the FRET efﬁciency between CoA 488 and ReAsH in 293T cells expressing dually tagged VRC01-IgM-BCR activated by V51 monomer antigen. Representative TIRFM images of V51-activated and non-activated 293T cells were shown. Cells with equal donor intensity and equal acceptor intensity were used for FRET analysis. Scale bar is 5 μm. Data are from at least 34 cells over five independent experiments. Q: quench; DQ: dequench. (**E–H**) FLIM-FRET to measure the FRET efﬁciency between CoA 488 and ReAsH of dually tagged VRC01-IgM-BCR expressed in 293T cells stimulated by V51 monomer antigen. (E) Representative FLIM images of V51-activated and non-activated 293T cells stained by donor only (CoA 488) or stained by donor and acceptor (CoA 488 + ReAsH) were shown. Scale bar is 3 μm. (**F–G**) Fluorescence lifetime and lifetime-based FRET efﬁciency of dually tagged VRC01-IgM-BCR expressing 293T cells. (H) FRET efﬁciency distribution of all measured dually tagged VRC01-IgM-BCR molecules. Data are from at least 11 cells. (I) Dequenching FRET to measure the FRET efﬁciency between CoA 488 and ReAsH in A20II1.6 B cells expressing dually tagged VRC01-IgM-BCR activated by V51 monomer antigen. Cells with equal donor intensity and equal acceptor intensity were used for FRET analysis. Data are from at least 28 cells. (J) The association of FRET efﬁciency between CoA 488 and ReAsH with the mean intensity of the p-Igα recruited into the immunological synapse in A20II1.6 B cells was indicated by Spearman's rank correlation coefficient (r). Error bars represent mean ± SEM. Two-tailed t-tests were used for the statistical comparisons. ***p<0.001; **p<0.01.

Figure 2—figure supplement 1. — (A) Schematic illustration showing FRET efﬁciency between CoA 488 label at N-terminus (red circle) and ReAsH label at Cμ2 domain (brown circle) of dually tagged VRC01-IgM-BCR. (B) Schematic illustration showing V51 monomer fused with poly-histidine tag presented on the surface of Ni²⁺-containing planar lipid bilayer (PLB) membranes on supported glass coverslips. (**C–D**) Dequenching FRET to measure the FRET efﬁciency between CoA 488 and ReAsH in 293T cells expressing dually tagged VRC01-IgM-BCR activated by V51 monomer antigen. Representative TIRFM images of V51-activated and non-activated 293T cells were shown. Cells with equal donor intensity and equal acceptor intensity were used for FRET analysis. Scale bar is 5 μm. Data are from at least 34 cells over five independent experiments. Q: quench; DQ: dequench. (**E–H**) FLIM-FRET to measure the FRET efﬁciency between CoA 488 and ReAsH of dually tagged VRC01-IgM-BCR expressed in 293T cells stimulated by V51 monomer antigen. (E) Representative FLIM images of V51-activated and non-activated 293T cells stained by donor only (CoA 488) or stained by donor and acceptor (CoA 488 + ReAsH) were shown. Scale bar is 3 μm. (**F–G**) Fluorescence lifetime and lifetime-based FRET efﬁciency of dually tagged VRC01-IgM-BCR expressing 293T cells. (H) FRET efﬁciency distribution of all measured dually tagged VRC01-IgM-BCR molecules. Data are from at least 11 cells. (I) Dequenching FRET to measure the FRET efﬁciency between CoA 488 and ReAsH in A20II1.6 B cells expressing dually tagged VRC01-IgM-BCR activated by V51 monomer antigen. Cells with equal donor intensity and equal acceptor intensity were used for FRET analysis. Data are from at least 28 cells. (J) The association of FRET efﬁciency between CoA 488 and ReAsH with the mean intensity of the p-Igα recruited into the immunological synapse in A20II1.6 B cells was indicated by Spearman's rank correlation coefficient (r). Error bars represent mean ± SEM. Two-tailed t-tests were used for the statistical comparisons. ***p<0.001; **p<0.01.

Distinguishing intermolecular FRET and intramolecular FRET

In all the above FRET experiments, some of the FRET signals might result from inter-complex juxtaposition of different BCRs since BCR oligomerization will occur upon antigen engagement. To minimize the involvement of FRET between neighboring BCRs, we thus only analyzed a subset of cells that had equivalent mean fluorescent intensity of donor and acceptor (i.e. equal density of dually tagged BCRs) in all experiments (Figure 2D,I). To further exclude these potential effects from intermolecular FRET, we similarly performed FRET experiments in 293T cells expressing dually tagged VRC01-IgM-BCR or two types of singly tagged VRC01-IgM-BCR. Plasmids carrying dually tagged VRC01-IgM-BCRs were diluted with plasmids carrying untagged BCRs and expressed in 293T cells to generate only intramolecular FRET within one individual heavy chain of mIg (Figure 2—figure supplement 1A), while plasmids carrying two types of singly tagged VRC01-IgM-BCRs (ybbR tag and tetracysteine tag are in different BCRs, respectively) were diluted with untagged BCRs and expressed in 293T cells to serve as a negative control presenting potential intermolecular FRET (Figure 2—figure supplement 1B), which might include a fraction of interchain FRET (FRET between CoA 488 and ReAsH labeled in two distinct heavy chains within one individual VRC01-IgM-BCR). Under the same experimental condition as above, antigen binding caused significant lower FRET between CoA 488 and ReAsH in cells expressing dually tagged VRC01-IgM-BCRs (Figure 2—figure supplement 1C). Meanwhile, FRET signal between CoA 488 and ReAsH in singly tagged VRC01-IgM-BCRs expressing cells was very low, and no FRET change was observed between these two fluorophores upon antigen engagement (Figure 2—figure supplement 1D), suggesting the alteration of FRET between N terminus and Cμ2 domain within IgM heavy chain generated by antigen-BCR interaction was mainly attributed by the increase of intramolecular distance between these two sites, rather than the intermolecular distance changes. We also compared the intramolecular FRET and intermolecular FRET between CoA 488 and ReAsH in 293T cells expressing non-diluted tagged VRC01-IgM-BCRs. Dually tagged VRC01-IgM-BCRs without dilution were expressed in 293T cells to generate intramolecular FRET (with a fraction of intermolecular FRET), while two types of singly tagged RC01-IgM-BCRs without dilution were expressed in 293T cells to generate intermolecular FRET. We found that intermolecular FRET accounted for about 1/3 of the detected intramolecular FRET signal, and antigen engagement significantly decreased intramolecular FRET, but not intermolecular FRET (Figure 2—figure supplement 1E,F).

Antigen-binding induced conformational change within mIg heavy chain is closely correlated to the strength of B cell activation

To assess whether antigen-binding induced conformational change within mIgM heavy chain is functionally relevant, we examined the association of the level of synaptic recruitment of phosphorylated Igα (p-Igα) with FRET efficiency between N terminus and Cμ2 domain in dually tagged VRC01-IgM-BCR expressing A20II1.6 B cells upon antigen stimulation. Inverse correlation between the FRET efficiency and the mean fluorescent intensity (mFI) of p-Igα was observed (Figure 2J), indicating the spatial distance between N terminus and Cμ2 domain within mIg is closely associated with the strength of B cell activation. However, it should be noted that another possible explanation for this association is that decreased FRET signal could be a consequence of more efficient antigen binding of the cell, thus enhancing the recruitment of p-Igα.

Conformational change within mIg heavy chain is specific to membrane IgM-BCR

Since the crystal structures of antigen-antibody complex indicate that antigen binding does not transmit conformational changes to the membrane-proximal regions of Ig molecule (Metzger, 1974; Metzger, 1992), we compared the conformational change within Ig of soluble VRC01-IgM and VRC01-IgM-BCR expressed on cell membrane. We expressed and purified dually tagged soluble VRC01-IgM monomers (Figure 1—figure supplement 2A,B), which can normally bind with gp120 antigen expressed on cells (Figure 1—figure supplement 2C) and can be labeled site-specifically (Figure 1—figure supplement 2D). Through acceptor photobleaching FRET method, we found no FRET change between N terminus and Cμ2 domain within soluble VRC01-IgM upon antigen binding (Figure 2—figure supplement 2A–C). Similarly, using FLIM-FRET method, no change of spatial distance between these two sites in soluble VRC01-IgM after antigen engagement was observed (Figure 2—figure supplement 2D–G). Therefore, data generated by different FRET detection methods demonstrated the decreased proximity between N terminus and Cμ2 domain within extracellular domains of IgM-BCR upon antigen stimulation, while no such change of spatial distance occurs between N terminus and Cμ2 domain within soluble IgM.

IgM- and IgG-BCR exhibited distinct conformational change pattern within the mIg heavy chain upon antigen binding

Given that the activation of the IgM- and IgG-BCR exhibited different mechanical force sensitivity (Wan et al., 2015), which might be correlated to the different conformational change within the mIg heavy chain, we thus investigated the antigen binding-induced FRET changes in the mIg heavy chain of IgM- versus IgG-BCR (Figure 3A). In comparison with IgM-BCR, distance between N terminus and Cγ2 domain in VRC01-IgG-BCR remained unchanged after interacting with V51 monomer antigen, as illustrated by the unaltered FRET between CoA 488 label at the N terminus and ReAsH label at Cγ2 of IgG heavy chain (Figure 3B, Figure 3—figure supplement 1A,B).

Figure 3. — (A) Schematic illustration of different types of VRC01-BCR equipped with different cytoplasmic tail. FRET efﬁciency between CoA 488 label at N-terminus (red circle) and ReAsH at Cμ2 domain (or Cγ2 portion, brown circle) of dually tagged VRC01-BCR was examined. (B) Dequenching FRET to measure the FRET efﬁciency between CoA 488 and ReAsH in 293T cells expressing dually tagged VRC01-BCR activated by V51 monomer antigen. From left to right: IgM, MMG, IgG, GGM. Error bars represent mean ± SEM. Data are from at least 19 cells over two independent experiments. Two-tailed t-tests were used for the statistical comparisons. ***p<0.001.

Figure 3—figure supplement 1. — (A) Schematic illustration of different types of VRC01-BCR equipped with different cytoplasmic tail. FRET efﬁciency between CoA 488 label at N-terminus (red circle) and ReAsH at Cμ2 domain (or Cγ2 portion, brown circle) of dually tagged VRC01-BCR was examined. (B) Dequenching FRET to measure the FRET efﬁciency between CoA 488 and ReAsH in 293T cells expressing dually tagged VRC01-BCR activated by V51 monomer antigen. From left to right: IgM, MMG, IgG, GGM. Error bars represent mean ± SEM. Data are from at least 19 cells over two independent experiments. Two-tailed t-tests were used for the statistical comparisons. ***p<0.001.

In addition, our previous study showed that the low mechanical force threshold of IgG-BCR activation is dependent on its cytoplasmic tail. Mechanistically, IgG cytoplasmic tail lowers the mechanical force threshold of IgG-BCR activation by enriching phosphatidylinositol (4,5)-biphosphate (PI(4,5)P2) into IgG-BCR membrane microdomains and potentially interacting with plasma membrane through its positively charged residues (Wan et al., 2015; Wan et al., 2018). This finding drove us to speculate that mIg cytoplasmic tail might be responsible for the distinct conformational change within the mIg heavy chain between IgM- and IgG-BCR in response to antigen binding. To determine the effect of mIg cytoplasmic tail on conformational change within mIg, we constructed VRC01-IgM-BCR equipped with a mIgG cytoplasmic tail (termed MMG-BCR thereafter) and VRC01-IgG-BCR equipped with a mIgM cytoplasmic tail (termed GGM-BCR thereafter), both of which possess one ybbR tag at N terminus of mIg heavy chain as VRC01-IgM- and VRC01-IgG-BCR (Figure 3A). In all constructs, FRET acceptor chromophore was ReAsH labeled at Cμ2 or Cγ2 region of mIg. The results showed that GGM-BCR exhibited the similar FRET change after antigen stimulation as IgM-BCR. In contrast, no significant conformational change occurred in antigen-stimulated MMG-BCR, resembling the behavior of IgG-BCR (Figure 3B, Figure 3—figure supplement 1A,B). In addition, we found that IgG cytoplasmic tail slightly decreased the basal FRET efficiency between CoA 488 and ReAsH in VRC01-BCR without antigen-binding (Figure 3—figure supplement 1C,D), while the difference was not significant. Moreover, two-way ANOVA showed a significant interaction effect between Ig cytoplasmic tail and antigen engagement on FRET between N terminus and Cμ2 (or Cγ2), indicating that Ig cytoplasmic tail significantly affected the antigen-induced conformational change within mIg of BCR. Two-way ANOVA with Sidak’s correction for multiple comparisons confirmed that antigen-binding significantly increased distance between N terminus and Cμ2 (or Cγ2) within mIg only in the absence of IgG cytoplasmic tail, while the presence of IgG cytoplasmic tail did not significantly increase the basal distance between these two sites of BCR without antigen-binding (Figure 3—figure supplement 2). All these results suggested that IgM- and IgG-BCR exhibit distinct conformational change within the mIg heavy chain upon antigen binding and such difference is dependent on the cytoplasmic tail of mIg.

Site-specific labeling of the extracellular domain of Igβ in BCR complex

It is assumed that the antigen engagement of mIg induces a conformational change within BCR complex to initiate recruitment of Lyn and phosphorylation of ITAMs at the cytoplasmic domain of Igα/β, facilitating the assembly of the membrane proximal signalosome in B cell. Thus, it is an intriguing hypothesis that the antigen-induced conformational change within mIg shall be somehow transmitted to Igα/β heterodimer, causing a further conformational change in the spatial relationship between mIg molecule and Igα/β within the BCR complex.

For the detection of intermolecular FRET between mIg and Igα/β heterodimer, we firstly tried to site-specifically label Fc region of mIg or Igα/β by ybbR tag insertion but failed. The difficulty of labeling might be caused by the potential steric hindrance effect between mIg and Igα/β (Radaev et al., 2010). Therefore, to measure FRET between mIg and Igα/β, tetracysteine tag was introduced into the extracellular domain of Igβ in both IgM- and IgG-BCR, and Fab fragment of antibody was used to label the Fc portion of mIg heavy chain (Figure 4—figure supplement 1A, Figure 4—figure supplement 2A) for measurement of FRET between mIg and Igβ. Confocal microscopy of transfected 293T cells showed that VRC01-BCRs with tetracysteine tag in Igβ were expressed and site-specifically labeled (Figure 4—figure supplement 1B, Figure 4—figure supplement 2B). Both WT and tagged VRC01-BCR can bind with V51 monomer (Figure 4—figure supplement 1C, Figure 4—figure supplement 2C). V51 trimer can induced calcium influx in tagged VRC01-BCR expressing A20II1.6 B cells, although the calcium signals in tagged VRC01-IgM-BCR expressing A20II1.6 B cells showed a little less sustained signal than cells expressing untagged VRC01-IgM-BCR (Figure 4—figure supplement 1D, Figure 4—figure supplement 2D). All these results indicated that VRC01-BCR tolerates the tetracysteine tag inserted in Igβ.

IgM- and IgG-BCR exhibited distinct conformational change pattern in the spatial relationship between mIg and Igβ upon antigen binding

We measured FRET efficiency between Alexa Fluor 647 label at Fc5μ fragment within IgM heavy chain and ReAsH label in Igβ of VRC01-IgM-BCR in 293T cells (Figure 4—figure supplement 3A). The result showed that no FRET change between these two sites occurred upon antigen binding, suggesting no change of spatial distance occurred between mIg and Igβ in IgM-BCR complex (Figure 4—figure supplement 3B,C).

Considering that IgM- and IgG-BCR showed distinct conformational change within mIg heavy chain after antigen engagement as aforementioned, we also examined the distance change between mIg and Igβ in antigen-activated VRC01-IgG-BCR (Figure 4A). We found a decreased intermolecular FRET efficiency between Alexa Fluor 647 label at Fcγ fragment of IgG heavy chain and ReAsH label in extracellular domain of Igβ measured by acceptor photobleaching FRET method in 293T cells (Figure 4B–C), indicating an increased spatial distance between mIg heavy chain and Igβ in VRC01-IgG-BCR upon the stimulation by membrane antigen. Similarly, cells with equal donor intensity and acceptor intensity were analyzed (Figure 4C). As a further validation, FLIM-FRET measurement confirmed that membrane V51 monomer antigen similarly caused a FRET reduction between these two sites in VRC01-IgG-BCR (Figure 4D–G). Increased distance between mIg heavy chain and Igβ was also confirmed in tagged VRC01-IgG-BCR expressing A20II1.6 B cells after binding with antigen (Figure 4H). Moreover, FRET efficiency between these two sites was found to be inversely correlated with the level of synaptic recruitment of p-Igα (Figure 4I), indicating the spatial distance between extracellular domains of mIg and Igβ is closely associated with the strength of B cell activation.

Figure 4. — (A) Schematic illustration showing FRET efﬁciency between ReAsH label in Igβ (brown circle) and AF647 label in Fc region (purple circle) of tagged VRC01-IgG-BCR. (**B–C**) Dequenching FRET to measure the FRET efﬁciency between ReAsH and AF647 in 293T cells expressing tagged VRC01-IgG-BCR activated by V51 monomer antigen. Representative TIRFM images of V51-activated and non-activated 293T cells were shown. Cells with equal donor intensity and equal acceptor intensity were used for FRET analysis. Scale bar is 5 μm. Data are from at least 30 cells over two independent experiments. Q: quench; DQ: dequench. (**D–G**) FLIM-FRET to measure the FRET efﬁciency between ReAsH and AF647 of tagged VRC01-IgG-BCR expressed in 293T cells stimulated by V51 monomer antigen. (D) Representative FLIM images of V51-activated and non-activated 293T cells stained by donor only (ReAsH) or stained by donor and acceptor (ReAsH +AF647) were shown. Scale bar is 3 μm. (**E–F**) Fluorescence lifetime and lifetime-based FRET efﬁciency of tagged VRC01-IgG-BCR expressing 293T cells. (G) FRET efﬁciency distribution of all measured tagged VRC01-IgG-BCR molecules. Data are from at least nine cells. (H) Dequenching FRET to measure the FRET efﬁciency between ReAsH and AF647 in A20II1.6 B cells expressing tagged VRC01-IgG-BCR activated by V51 monomer antigen. Cells with equal donor intensity and equal acceptor intensity were used for FRET analysis. Data are from at least 24 cells. (I) The association of FRET efﬁciency between ReAsH and AF647 with the mean intensity of the p-Igα recruited into the immunological synapse in A20II1.6 B cells was indicated by Spearman's rank correlation coefficient (r). Error bars represent mean ± SEM. Two-tailed t-tests were used for the statistical comparisons. ***p<0.001; **p<0.01; *p<0.05.

Figure 4—figure supplement 1. — (A) Schematic illustration showing FRET efﬁciency between ReAsH label in Igβ (brown circle) and AF647 label in Fc region (purple circle) of tagged VRC01-IgG-BCR. (**B–C**) Dequenching FRET to measure the FRET efﬁciency between ReAsH and AF647 in 293T cells expressing tagged VRC01-IgG-BCR activated by V51 monomer antigen. Representative TIRFM images of V51-activated and non-activated 293T cells were shown. Cells with equal donor intensity and equal acceptor intensity were used for FRET analysis. Scale bar is 5 μm. Data are from at least 30 cells over two independent experiments. Q: quench; DQ: dequench. (**D–G**) FLIM-FRET to measure the FRET efﬁciency between ReAsH and AF647 of tagged VRC01-IgG-BCR expressed in 293T cells stimulated by V51 monomer antigen. (D) Representative FLIM images of V51-activated and non-activated 293T cells stained by donor only (ReAsH) or stained by donor and acceptor (ReAsH +AF647) were shown. Scale bar is 3 μm. (**E–F**) Fluorescence lifetime and lifetime-based FRET efﬁciency of tagged VRC01-IgG-BCR expressing 293T cells. (G) FRET efﬁciency distribution of all measured tagged VRC01-IgG-BCR molecules. Data are from at least nine cells. (H) Dequenching FRET to measure the FRET efﬁciency between ReAsH and AF647 in A20II1.6 B cells expressing tagged VRC01-IgG-BCR activated by V51 monomer antigen. Cells with equal donor intensity and equal acceptor intensity were used for FRET analysis. Data are from at least 24 cells. (I) The association of FRET efﬁciency between ReAsH and AF647 with the mean intensity of the p-Igα recruited into the immunological synapse in A20II1.6 B cells was indicated by Spearman's rank correlation coefficient (r). Error bars represent mean ± SEM. Two-tailed t-tests were used for the statistical comparisons. ***p<0.001; **p<0.01; *p<0.05.

Since the cytoplasmic tail of mIg molecule was found to be involved with the mechanical force requirement of BCR activation (Wan et al., 2015; Wan et al., 2018) and was related with the conformational change within extracellular domains of mIg in BCR complex upon antigen engagement, as described in our published study and abovementioned result, respectively, we thus investigated the effect of mIg cytoplasmic tail on antigen-induced conformational change in the spatial relationship between mIg molecule and Igβ (Figure 5A). In line with the change observed in antigen-stimulated VRC01-IgG-BCR, spatial distance between mIg heavy chain and Igβ in IgG cytoplasmic tail carrying VRC01-MMG-BCR increased upon antigen engagement, as illustrated by the decreased FRET efficiency. By contrast, VRC01-GGM-BCR possessing IgM cytoplasmic tail resembled the behavior of VRC01-IgM-BCR, in which the distance between mIg and Igβ remained unaffected after antigen activation (Figure 5B, Figure 5—figure supplement 1A,B). In addition, we observed that the presence of IgG cytoplasmic tail significantly increased the basal FRET efficiency between ReAsH and AF647 in VRC01-BCR without antigen binding (Figure 5—figure supplement 1C,D). Moreover, two-way ANOVA showed a significant interaction effect between Ig cytoplasmic tail and antigen engagement on FRET between mIg and Igβ, indicating that Ig cytoplasmic tail significantly affected the antigen-induced conformational change in the spatial relationship between mIg and Igβ. Two-way ANOVA with Sidak’s correction for multiple comparisons confirmed that antigen-binding significantly increased the distance between mIg and Igβ only in the presence of IgG cytoplasmic tail, while IgG cytoplasmic tail significantly decreased the basal distance between these two molecules (Figure 5—figure supplement 2).

Figure 5. — (A) Schematic illustration of different types of VRC01-BCR equipped with different cytoplasmic tail. FRET efﬁciency between ReAsH label at extracellular domain of Igβ (brown circle) and AF647 label at Fc portion (purple circle) of tagged VRC01-BCR was detected. (B) Dequenching FRET to measure the FRET efﬁciency between ReAsH and AF647 in 293T cells expressing tagged VRC01-BCR activated by V51 monomer antigen. From left to right: IgM, MMG, IgG, GGM. Error bars represent mean ± SEM. Data are from at least 19 cells over two independent experiments. Two-tailed t-tests were used for the statistical comparisons. **p<0.01.

Figure 5—figure supplement 1. — (A) Schematic illustration of different types of VRC01-BCR equipped with different cytoplasmic tail. FRET efﬁciency between ReAsH label at extracellular domain of Igβ (brown circle) and AF647 label at Fc portion (purple circle) of tagged VRC01-BCR was detected. (B) Dequenching FRET to measure the FRET efﬁciency between ReAsH and AF647 in 293T cells expressing tagged VRC01-BCR activated by V51 monomer antigen. From left to right: IgM, MMG, IgG, GGM. Error bars represent mean ± SEM. Data are from at least 19 cells over two independent experiments. Two-tailed t-tests were used for the statistical comparisons. **p<0.01.

In summary, these results suggested that the antigen-induced conformational change within mIg heavy chain might be transmitted to Igα/β, decreasing proximity between mIg and Igβ of IgG-BCR. In addition, conformational change in the spatial relationship between mIg and Igβ is dependent on the cytoplasmic tail of mIg and is associated with the initial intracellular signaling in activated B cell.

Discussion

B cell activation is triggered by BCR-antigen engagement. When BCRs encounter antigens, the mobility of BCRs will be reduced followed by BCR oligomerization and the formation of BCR microclusters (Tolar et al., 2005; Harwood and Batista, 2010; Mattila et al., 2013; Liu et al., 2010a; Liu et al., 2010b). BCR is an extraordinary receptor that can efficiently discriminate the biochemical and biophysical features of an antigen, such as the stiffness of the substrates presenting the antigen (Wan et al., 2013; Zeng et al., 2015; Shaheen et al., 2017; Wang et al., 2018b) and mechanical force signals delivered by antigen (Wan et al., 2015; Wan et al., 2018; Natkanski et al., 2013; Nowosad et al., 2016; Spillane and Tolar, 2017). However, it remains an open but fundamental question in terms of the molecular mechanism of how the BCR transduces antigen signals. For example, it is still elusive of how the extracellular antigen binding signal is transduced to the cytoplasmic domain of BCR complex to trigger downstream signaling cascades?

In the field, it has been proposed that the conformational change model of BCR might be applicable in the BCR-mediated initiation of B cell activation, mirroring the similar model of T cell receptor (TCR) activation (Gil et al., 2002; Lee et al., 2015; Ma et al., 2008; Xu et al., 2008; Blanco et al., 2014; Swamy et al., 2016; Schamel et al., 2017; Dustin and Davis, 2014; Depoil and Dustin, 2014; Dustin, 2009). However, several crystal structures of antibodies in the presence of soluble antigens indicate that antigen binding seems incapable of propagating significant conformational changes to membrane-proximal regions of Ig (Davies and Metzger, 1983; Davies et al., 1990; Metzger, 1978). Our results in this report also confirmed that no conformational change occurs within soluble VRC01-IgM molecule upon membrane antigen engagement via FRET measurement between N terminus and Cμ2 region. We shall emphasize that there is a significant difference between the conditions in which the characterization of antigen-antibody complexes was examined and those under which we explored the antigen-BCR interaction on cell plasma membrane. Indeed, several studies directly focused on conformational changes within intracellular domains of BCR (Tolar et al., 2005; Lee and Tolar, 2013; Chen et al., 2015) suggest the lack of structural-based evidence at this stage does not preclude the possibility that conformational changes may contribute to the initiation of B cell activation. In addition, according to our published studies, the interaction between cytoplasmic tail and cell membrane is necessary for the antigen-induced conformational change in cytoplasmic domain of BCR (Chen et al., 2015). It is also reported that extracellular conformational change-mediated oligomerization of the BCR requires the WTxxST motif in the transmembrane region of mIg (Tolar et al., 2009), which is lacked in soluble Ig. These findings indicate that interaction between BCR complex and cell membrane might be crucial for conformational changes within ectodomains of BCR upon antigen engagement. Nevertheless, whether conformational changes occur at extracellular domains of BCR remains unknown. Investigation on this process will facilitate us to understand how BCR transmit extracellular antigen signal across the membrane to the intracellular component to trigger downstream signaling during early B cell activation, providing us molecular mechanism for fundamentals of B cell responses. In the present study, we combined site-specific labeling methodology and FRET-based assay to address above-mentioned questions. By inserted short peptides that allow targeted incorporation of fluorophores into different regions, we achieved site-specific labeling within BCR complex to enable monitoring distance change between two labeled sites through FRET. We checked two FRET pairs: one pair represented conformational change within mIg heavy chain, whereas the other pair demonstrated conformational change in the spatial relationship between mIg and Igα/β. We also compared the conformational change between antigen-bound IgM- and IgG-BCR.

We firstly found decreased FRET efficiency between N terminus and Cμ2 domain within mIg of IgM-BCR upon antigen engagement. Notably, decreased proximity between this two sites were discovered in both 293T cells and A20II1.6 B cells, indicating the independence of the conformational change on downstream signaling after antigen stimulation. Since these two regions locate across the hinge region linking Fab and Fc fragment of mIg, distance change between N terminus and Cμ2 domain observed in this study might involve stretching or bending of the hinge region. Interestingly, the level of conformational change (as indicated by the FRET efficiency) within mIg of IgM-BCR was positively associated with the strength of B cell activation. However, such conformational change was not observed in IgG-BCR. We also found that conformational change within mIg was dependent on the absence of IgG cytoplasmic tail, while a reproducible trend was consistently observed that the presence of IgG cytoplasmic tail slightly, but non-significantly increased the basal distance between N terminus and Cμ2 (or Cγ2) domain of VRC01-BCR without antigen binding. Combining all these findings, we speculated that in the absence of IgG cytoplasmic tail, antigen engagement induced conformational change in the spatial relationship between N terminus and Cμ2 domain within mIg of IgM-BCR, while greater conformational change was associated with higher activation level in B cell. Potentially, conformational change in the spatial relationship between N terminus and Cμ2 domain within mIg of antigen-bound IgM-BCR was then transmitted to membrane-proximal domain, while this transduction was achieved in IgG-BCR without change of spatial distance between N terminus and Cγ2 domain. Extracellular antigen signal transmitted to membrane-proximal region might trigger local conformational change in Fc domain of mIg, inducing an orientation to expose a clustering interface on the Cμ4 (or Cγ3) domain (Tolar et al., 2009) (Figure 6).

Figure 6. — (A) Conformational change model of IgM-BCR activation. Upon antigen engagement, distance between N-terminus and Cμ2 domain within mIgM increases, which might be resulted from stretching or bending of the hinge region. This conformational change might trigger the potential conformational change within Fc domain to expose the clustering interface in Cμ4 domain. (B) Conformational change model of IgG-BCR activation. After binding with antigen, potential conformational change within Cγ3 domain occurs independently of the distance change between N-terminus and Cγ2 within mIgG. Then the proximity between mIg and Igα/β decreases, which might be involved with the dissociation between IgG cytoplasmic tail plasma membrane. The exposure of the clustering interface in Cμ4 (or Cγ3) domain will then promote BCR clustering.

In our experimental system, intermolecular FRET occurred between neighboring BCR complexes as BCR cluster formation might interfere the FRET signals between interested areas within one BCR complex, although according to the research in TCR, intermolecular FRET was very low (Lee et al., 2015). To solve this, when exploring the conformational change within BCR engaged with membrane antigen, we analyzed data from cells with equivalent mean density of donor and acceptor (indicated by mFI). We also deconvolved the intramolecular FRET and intermolecular FRET by expressing untagged BCRs to dilute tagged BCRs in cells, proving the FRET change mainly resulted from intramolecular FRET, rather than intermolecular FRET.

Antigen binding leads to phosphorylation of ITAMs in Igα/β heterodimer, which is noncovalently linked with mIg molecule. In the proposed conformational change model of BCR-mediated B cell activation, signals generated from extracellular stimulation will finally reach at Igα/β through mIg component. We discovered decreased FRET efficiency between Fc region of mIg and Igβ upon antigen engagement in IgG-BCR expressing 293T cells and A20II1.6 B cells. Similarly, conformational change in the spatial relationship between these two sites was also dependent on mIg cytoplasmic tail (Figure 6). Interestingly, we found that in VRC01-BCR without antigen binding, the presence of IgG cytoplasmic tail increased the basal proximity between mIg and Igβ, indicating IgG cytoplasmic tail might affect the interaction between mIg and Igβ in the absence of antigen. This effect of IgG cytoplasmic tail might involve its interaction with the inner leaflet of the plasma membrane (Chen et al., 2015; Wan et al., 2018). Furthermore, the extent of conformational change in the spatial relationship between mIg and Igβ was positively associated with the B cell activation level. Increased spatial distance between mIg and Igα/β of IgG-BCR might generated from the dissociations of IgG cytoplasmic tail and Igα from the plasma membrane (Lee and Tolar, 2013; Chen et al., 2015) (Figure 6B). Meanwhile, it has been reported that the extracellular domains of Igα/β preferentially recognize the Fc region of IgM compared with IgG (Radaev et al., 2010), potentially inhibit the dissociation between mIg and Igβ in IgM-BCR. In line with previous findings, reduced proximity between extracellular domains of mIg and Igβ might induce the increased distance between cytoplasmic domains of mIg and Igα/β heterodimer, facilitating the formation of ‘umbrella opening form’ in cytoplasmic domains of antigen-activated BCR (Tolar et al., 2005).

One limitation of our study is that the physiological meanings of the observed conformational changes within extracellular domain of BCR remain unclear. Although the association between conformational changes of BCR and the strength of B cell activation was found, it is still premature to conclude that the antigen-induced conformational changes of BCR are causal for triggering downstream signaling of B cell. The actual role (activating or inhibitory effect) of the reported BCR conformational changes in B cell activation can be complex depending on the context of the antigens, the concept of which can be reflected from the investigations on conformational changes of other membrane receptors, such as G-protein-coupled receptor (GPCR). Agonist and antagonist will induce distinct conformational change of GPCR, indicating different conformational changes within GPCR might trigger different physiological functions (Vafabakhsh et al., 2015; Olofsson et al., 2014). Specifically, the observation that conformational change between N terminus and Cμ2 domain within mIg of IgM-BCR (which was positively associated with the B cell activation level) did not occur in the presence of IgG cytoplasmic tail seems to contradict with the previous findings, which report the enhancement of IgG cytoplasmic tail on the antigen-induced B cell activation (Chen et al., 2015; Liu et al., 2010b; Wakabayashi et al., 2002). One possible explanation is that conformational change in the spatial relationship between N terminus and Cμ2 (or Cγ2) domain is only required for IgM-BCR (or IgG-BCR equipped with IgM cytoplasmic tail) to transmit antigen-binding signal to membrane-proximal domain, inducing an orientation to expose a clustering interface on the Cμ4 (or Cγ3) domain (Tolar et al., 2009). In contrast, this conformational change is probably not required for IgG-BCR (or IgM-BCR equipped with IgG cytoplasmic tail) to transmit the extracellular signal of antigen-binding to induce the potential conformational change near the Cγ3 (or Cμ4) domain. This hypothesis seems to be consistent with the previous findings that IgG cytoplasmic tail lowers the threshold of mechanical force to induce IgG-BCR activation (Wan et al., 2015; Wan et al., 2018). Considering that the mechanical force might be closely related with the conformational change, it is reasonable to speculate that IgG cytoplasmic tail might lower the threshold of B cell activation through bypassing the required conformational change between N terminus and Cμ2 domain within mIg in IgM-BCR. Thus, it will be constructive to investigate the association between mechanical force and conformational change of BCR in the future study. Another possible explanation is that spatial distance change between N terminus and Cμ2 domain of mIg in IgM-BCR could be interpreted as an inhibitory mechanism. Antigen-induced conformational change between N terminus and Cμ2 domain might inhibit further BCR activation through an unexplored mechanism. An interesting hypothesis is that distance change between N terminus and Cμ2 domain might affect the BCR-antigen bond lifetime. As accumulation of bond lifetimes of TCR-pMHC is required for T cell signaling according to a published study (Liu et al., 2014), it shall be intriguing to explore the effect of antigen-induced conformational change on BCR-antigen bond lifetime.

The mechanism by which IgG cytoplasmic tail influences the antigen-induced conformational changes of BCR is also worth further exploration. We speculate that through its interaction with cell membrane (Chen et al., 2015), IgG cytoplasmic tail might alter the basal conformational state of BCR complex (in fact, we found that it can significantly affect the distance between mIg and Igα/β in the absence of antigen), or alter the mechanical force sensing capability of BCR (Wan et al., 2018). Analyzing the structure of BCR complex by cryo-electron microscopy might uncover the exact mechanism.

In summary, this study reports antigen-induced conformational changes within extracellular domain of BCR, providing evidence for the ‘conformational change model’, which has been proposed as a possible mechanism to explain the initiation of antigen-induced B cell activation. Furthermore, these conformational changes are associated with BCR-mediated B cell signaling. These observations may offer a perspective for the further investigation of triggering and regulating mechanisms in BCR activation and B cell function. In addition, our study provides a framework for exploring ligand-induced intramolecular or intermolecular events in BCR and other immune receptors.

Materials and methods

Plasmids, cells, antibodies and reagents

Plasmids carrying components of BCRs were all constructed in pHAGE backbone. In plasmids carrying tagged mIg heavy chains, ybbR tag (DSLEFIASKLA) and/or tetracysteine tag (FLNCCPGCCMEP) were inserted. In plasmid carrying tagged Igβ, tetracysteine tag was inserted. Insertion sites for the ybbR tag or tetracysteine tag are indicated in bold in the following table.

Construct	Amino acid sequence
ybbR tag in N terminus of VRC01-BCR	DSLEFIASKLAQVQLVQSGGQ
tetracysteine tag in Cμ2 of VRC01-IgM-BCR	VPPRDGFFGNFLNCCPGCCMEPPRKSKLICQA
tetracysteine tag in Cγ2 of VRC01-IgG-BCR	KTHTCPPCPAFLNCCPGCCMEPPELLGGPSVF
tetracysteine tag in Igβ	IYFCQQKCNNFLNCCPGCCMEPTSEVYQGCGT

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Human IgM and IgG constant regions of heavy chains were fused with VRC01-specific variable region to construct mIg heavy chains of VRC01-IgM- and IgG-BCR, respectively. Plasmids carrying mutant Ig cytoplasmic tails of heavy chains were constructed based on the WT versions. Human Igκ constant region was fused with VRC01-specific variable region to construct mIg light chain of VRC01-BCR. A20II1.6 B cells or 293T cells were co-transfected with mIg heavy chain, mIg light chain, human Igα and human Igβ by electroporation (Lonza) or calcium phosphate transfection, respectively. A20II1.6 B cell line (Liu et al., 2010c) and Ramos B cell line (RRID:CVCL_0597) were gifts from Dr. Susan K. Pierce (NIAID, NIH, USA) that were originally purchased from ATCC (USA) and maintained in complete RPMI-1640 medium with 10% heat-inactivated fetal bovine serum (FBS), penicillin and streptomycin antibiotics (Invitrogen). 293T cells were purchased from Cell bank (Chinese academy of sciences, Shanghai) and cultured in DMEM containing 10% FBS, penicillin, and streptomycin antibiotics. V51 monomer and V51 trimer antigens were gifts from Dr. Zhang Linqi (Tsinghua University, Beijing, China). All cell lines used in this study were negative for mycoplasma contamination test based on PCR detection method. Goat non-specific IgG, Alexa Fluor 647 conjugated Fab anti-human IgM Fc5μ and Alexa Fluor 647 conjugated Fab anti-human IgG Fcγ were purchased from Jackson ImmunoResearch. CoA 488 and Sfp phosphopantetheinyl transferase (SFP synthase) used for ybbR tag labeling were purchased from NEB. ReAsH-EDT2 for tetracysteine tag labeling was purchased from Santa Cruz. Chemical reagents used in all experiments were purchased from Sigma Aldrich.

Purification of soluble VRC01-IgM

WT or tagged VRC01-IgM monomers (without J chain) fused with a His₆ tag at C terminus were expressed in 293 F cells. The culture supernatants were purified by Ni²⁺ column and were further purified by Superdex 200 column (GE Healthcare).

Site-specific labeling of BCR and soluble Ig

Labeling of ybbR tag in cells was achieved according to published protocols (Yin et al., 2006). Briefly, 293T or A20II1.6 B cells expressing ybbR-tagged BCR were incubated with 1 μM SFP synthase, 10 mM MgCl₂, 2 μM CoA 488 in HBSS buffer for 20 min at room temperature. For tetracysteine-tagged BCR labeling, cells were pre-incubated with 5 mM 2-mercaptoethanesulfonate (MES) and 0.5 mM tris(carboxyethyl)phosphine (TCEP) at 37°C for 20 min, then the pre-treated cells were stained in HBSS buffer containing 1 μM ReAsH-EDT2 and 25 μM 2,3-dimercapto-1-propanol (BAL) for 10 min at 4°C, then washed by 100 μM BAL (Hoffmann et al., 2010). For Fc5μ or Fcγ fragment labeling, cells were stained for 5 min at 4°C with Alexa Fluor 647 conjugated Fab anti-human IgM Fc5μ or Alexa Fluor 647 conjugated Fab anti-human IgG Fcγ, respectively. For ybbR tag labeling in soluble tagged soluble VRC01-IgM, purified proteins were incubated with 1 μM SFP synthase, 10 mM MgCl₂, 5 μM CoA 488, 10 mM HEPES for 30 min at 37°C. For ReAsH labeling, tagged soluble VRC01-IgM molecules were treated with 1 μM ReAsH-EDT2 in HBSS buffer for 30 min at room temperature. Labeled proteins were desalted using Zeba Spin Desalting Columns (Thermo Fisher).

Ca²⁺ mobilization analysis

To detect the intracellular Ca²⁺ mobilization of A20II1.6 B cells expressing tagged VRC01-BCR, cells were processed with 1 μM Fluo4-AM (Invitrogen) and 2.5 mM probenecid (Invitrogen) at 30 °C for 30 min in HBSS (with Ca²⁺ and Mg²⁺) buffer. Then cells were incubated at 37 °C for 15 min in HBSS buffer. Treated cells were stimulated with 100 nM HIV-1 gp120 antigen, calcium ﬂux was measured by BD Accuri C6 cytometer and analyzed by FlowJo software.

V51 monomer binding measurement

293T cells expressing WT or tagged VRC01-BCR were firstly incubated with 50 nM V51 monomer (fused with poly-histidine tag) for 10 min at room temperature followed by washing with PBS. Then cells were treated with anti-His tag antibody (mouse IgG1, Biodragon Immunotechnologies, Beijing) at room temperature for 10 min. After washed with PBS, cells were stained with Alexa Fluor 488-conjugated-affinipure goat anti-mouse IgG, Fcγ fragment specific (minimal cross-reaction to human, bovine, and horse serum proteins, Jackson ImmunoResearch) at 4°C for 10 min. Results were acquired by BD Accuri C6 cytometer and analyzed by FlowJo software.

Preparation of antigen-containing coverslip

Glass coverslips (VWR International) were pretreated with acid (H₂SO₄:H₂O₂ = 7:3) and then were washed and dried before glued to the 8-well chamber frame (Nunc Lab-Tek chamber). For experiments in which cells were stimulated with membrane V51 monomer antigen, 0.1 mM Ni²⁺-containing planar lipid bilayer (PLB) were prepared as our published study (Zhang et al., 2013) and loaded on coverslip for 30 min at room temperature followed by washing with PBS, then 20 nM V51 monomer (fused with poly-histidine tag) were added and incubated for 30 min at room temperature. After addition of antigen, chambered coverslip was washed with PBS and then was blocked with 1% casein in PBS for 30 min at 37°C. Finally, the labeled cells were loaded on the antigen surface for reaction at 37°C for 3 min and fixed with 4% paraformaldehyde (PFA). In control groups without antigen, coverslip was pre-treated with acid, after which Ni²⁺-PLB were added and incubated, then coverslip was blocked by 1% casein. Finally, the cells were loaded on the surface without V51 molecules.

For experiments in which soluble VRC01-IgM molecules were stimulated with membrane V51 monomer antigen, coverslip was similarly pre-treated with acid, incubated with Ni²⁺-PLB followed by V51 molecules, and blocked with 1% casein. Then the soluble VRC01-IgM molecules were added on the antigen surface. After reaction for 10 min at 37°C, soluble VRC01-IgM molecules were imaged by confocal fluorescence microscopy. In control groups without antigen, coverslip was pre-treated with acid and blocked with 1% casein. Then soluble VRC01-IgM molecules were loaded on coverslip without V51 molecules and were incubated to allow the non-specific adherence on the surface before imaging.

Molecular imaging by TIRFM and confocal fluorescence microscopy

TIRFM images were captured by an Olympus IX-81 microscope equipped with a TIRF port, an Olympus 100 × 1.49 NA oil objective lens, and an Andor iXon +DU-897D electron-multiplying charge-coupled device camera. Image acquisition was controlled by MetaMorph software (Molecular Devices) with a 100 ms exposure time for a 512 × 512 pixel image. Confocal images were acquired by a FLUOVIEW FV1000 confocal laser scanning microscope (Olympus) with a 60 × 1.42 NA oil objective lens. All the images were acquired while keeping the cells in HBSS and were analyzed with ImageJ (National Institutes of Health). The mFI of donor fluorophores, acceptor fluorophores and signaling molecules were calculated as described previously (Liu et al., 2010a; Liu et al., 2010b; Liu et al., 2010c). When acquiring images of resting cells without antigen binding, we selected cells with high fluorescent intensity to match resting cells to antigen-stimulated cells.

FRET measurement by acceptor photobleaching method

Images of stimulated and fixed cells on antigen-containing coverslip were acquired by TIRFM, while images of soluble Ig molecules were obtained by confocal fluorescence microscopy. FRET pairs used here included CoA 488 (donor) +ReAsH (acceptor), and ReAsH (donor) +Alexa Fluor 647 (acceptor). All the images were processed with ImageJ. FRET efficiency was calculated with the formula: FRET efficiency = (DQ − Q)/DQ, where DQ and Q representing dequenched and quenched donor fluorescence intensity, respectively. To match the fluorescent intensity of donor and acceptor in resting cells and antigen-stimulated cells for analysis, resting cells with relatively low fluorescent intensity and antigen-stimulated cells with relatively high fluorescent intensity were excluded.

FRET measurement by FLIM

Time-correlated single-photon counting (TCSPC) system (PicoQuant) was used for FLIM-FRET measurement following the published protocol (Sun et al., 2011; Jahn et al., 2015). Dually labeled (CoA 488 + ReAsH, ReAsH +Alexa Fluor 647) and donor only labeled (CoA 488, ReAsH) cells, or dually labeled (CoA 488 + ReAsH) and donor only labeled (CoA 488) soluble VRC01-IgM molecules in HBSS buffer were loaded to antigen-containing coverslip. FLUOVIEW FV1200 confocal fluorescence microscope consisting of an inverted microscope (IX 83, Olympus) equipped with an Olympus UPLSAPO 60 × NA 1.3 oil immersion objective was used to collect the fluorescence signal. The donor fluorophore (CoA 488 or ReAsH) was excited with 480 nm or 560 nm laser and the donor signal was detected with 520/35 nm or 615/60 nm bandpass filter, respectively. In all experiments, the laser power was adjusted to achieve average photon counting rates ≤ 10⁵ photons/s. All FLIM data acquisition and analysis were performed with SymPhoTime 64 software.

Intracellular immunofluorescence staining and molecular imaging

The recruitment of signaling molecules into the immunological synapse of B cells stimulated by membrane antigen was imaged by TIRFM by following our previously published protocol (Liu et al., 2010a; Liu et al., 2010b; Liu et al., 2010c). In brief, stimulated A20II1.6 B cells fixed on antigen-containing chambered coverslip were permeabilized with 0.1% Triton X-100 and then blocked with 0.1 mg/ml goat non-specific IgG. Subsequently, cells were stained with phosphor-CD79A (Tyr182) primary antibody (Cell Signaling Technology) at room temperature for 1 hr. After washing with PBS, cells were stained with secondary antibody Alexa Fluor 488 or 647-conjugated F(ab’)₂ goat anti-rabbit IgG H + L (Invitrogen) at room temperature for 45 min. After washing with PBS, the cells were imaged by TIRFM and analyzed by ImageJ.

Acknowledgements

We acknowledge the assistance of Image Core Facility, Technology Center for Protein Sciences, Tsinghua University, for assistance of using FV1200 LSCM with Picoquant FLIM/FCS system. This work is supported by funds from National Science Foundation China (81825010, 81730043, 81621002 and 31811540397).

Funding Statement

The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.

Contributor Information

Wanli Liu, Email: liulab@tsinghua.edu.cn.

Arup K Chakraborty, Massachusetts Institute of Technology, United States.

Michael L Dustin, University of Oxford, United Kingdom.

Funding Information

This paper was supported by the following grants:

National Natural Science Foundation of China 81825010 to Wanli Liu.
National Natural Science Foundation of China 81730043 to Wanli Liu.
National Natural Science Foundation of China 81621002 to Wanli Liu.
National Natural Science Foundation of China 31811540397 to Wanli Liu.

Additional information

Competing interests

No competing interests declared.

Author contributions

Conceptualization, Data curation, Validation, Investigation, Methodology, Writing—original draft, Writing—review and editing.

Investigation, Methodology, Writing—review and editing, Assisted with all experiments.

Investigation, Methodology, Writing—review and editing, Carried out protein expression and purification experiments.

Conceptualization, Writing—review and editing.

Investigation, Methodology, Assisted with protein labeling experiments.

Writing—review and editing.

Conceptualization, Resources, Supervision, Funding acquisition, Validation, Methodology, Project administration, Writing—review and editing.

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elife-42271-transrepform.docx^{(245.2KB, docx)}

DOI: 10.7554/eLife.42271.019

Data availability

All data generated or analysed during this study are included in the manuscript and supporting files.

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eLife. doi: 10.7554/eLife.42271.021

Decision letter

Editor: Michael L Dustin¹

Reviewed by: Pavel Tolar², Balbino Alarcon³

In the interests of transparency, eLife includes the editorial decision letter and accompanying author responses. A lightly edited version of the letter sent to the authors after peer review is shown, indicating the most substantive concerns; minor comments are not usually included.

Thank you for submitting your article "Conformational change of B cell receptor in the initiation of B cell activation" for consideration by eLife. Your article has been reviewed by Arup Chakraborty as the Senior Editor, a Reviewing Editor, and three reviewers. The following individuals involved in review of your submission have agreed to reveal their identity: Pavel Tolar (Reviewer #1); Balbino Alarcon (Reviewer #2).

The reviewers have discussed the reviews with one another and the Reviewing Editor has drafted this decision to help you prepare a revised submission.

The authors generate some intriguing data that suggest conformational changes of BCR that are induced by antigen on the cell surface, but there is some concern that the authors try to take on too many different questions related to this without establishing a strong enough foundation to analyze the FRET signatures. If the authors were to focus on the site-specific labeling system set up in VRC01 IgM and fully characterize it with all necessary controls, they could have a strong study that may provide an unprecedented picture IgM extracellular domain conformation change on binding of a monovalent antigen.

Essential revisions:

1) All reviewers were concerned about the use the polyclonal Fab in FRET studies of IgM and IgG. There are 6 Ig domains in IgM Fc and 4 in IgG Fc to which these reagents could bind. So, these reagents were considered inappropriate for the task in terms of both steric effects of Fab multiple binding and the uncertainty as to the location of binding if all sites were not saturated. Unfortunately, none of the experiments using these reagents will be appropriate for publication at the level eLife aspires to. On the other hand, the site specific labeling strategy in the VRCO1 BCR were considered sufficiently site specific and well characterized functionally to be of great use. The authors are urged to build a better foundation around VRC01 binding to gp120 monomers (verify). The reviewers have made some suggestions below regarding possible ways forward that might be achievable in a revised manuscript in 2 months, however, the authors are given latitude to take other approaches that provide interpretable FRET data from a well defined system to provide convincing evidence of conformational changes.

2) The authors should be able to generate CoA488 and ReAsH labeled soluble IgM and study the FRET in solution and the effect of antigen binding with absolute quantification. While this is an IgM antibody, there are ways to produce these in 293 cells without or with J-chain to generate monomers or pentamers. This would enable a systematic study of the conformational dynamics of the receptors in solution with and without ligand and binding and forced clustering. The system could be used to calibrate the FRET signature of cell surface FRET. These experiments could be done in bulk, single cell or microscopy modes.

3) The authors should verify that gp120 is monomeric and could determine the consequences of gp120 binding to the monomeric or pentameric soluble IgM in terms of any evidence for generating of higher order structures, which would suggest that the gp120 is not monomeric or can be induced to form oligomers when engaged by the Fab. But the resulting multimers could be characterized by gel filtration or analytical ultracentrifugation.

4) Parts of Figure 1, Figure 2Figure 6 and Figure 7 could be used as a core for a revised paper that provides a better-controlled study that focuses on the gp120 system. The authors may have sufficient time to generate results with solid phase gp120, which can be attached to a solid substrate or presented in supported lipid bilayers.

The revisions should also address:

1) Does the BCR undergo a conformational change detected in the ectodomains upon antigen binding? With soluble IgM they might hope to be able to model the FRET and even estimate average distances.

2) Does the conformational change correlate with downstream activation signaling pathways? The experiments with IgGa/b labeling in the gp120 system may shed light on this.

3) The site specific labeling of the BCR is a novel and useful tool. But to interpret the donor-dequenching data, it is important to know the labelling efficiency with the donor and acceptor. The relative stoichiometry of the labels is particularly critical for comparing the different conditions and BCR constructs. FLIM is a good way to determine the level of FRET irrespectively of labelling stoichiometry, and the authors do use it in one experiment, but unfortunately, they do not derive the information about the fraction of donor molecules in FRET and their FRET level. This could be achieved with soluble IgM and lessons applied to understanding what is happening on the cell surface.

4) Controls for the FRET photobleaching measurements using donor only and acceptor only samples were missing. These are important as there is often some bleedthrough and also some photobleaching-induced photoconversion. Given that the antigen-induced FRET changes are small, these additional corrections may be significant.

5) Even if the authors don't perform the calibration studies with soluble IgM, they need to deconvolve intermolecular and intramolecular FRET. Its felt that soluble monomers and pentamers will provide critical insight into this, but otherwise another method to look separate out these effects is needed.

6) The authors should provide a better structural model for the conformational changes implied by the FRET measurements. It seems that there are limited number of ways to explain the results and authors should be able to provide schematics to systematically explore these, which may clarify experiments needed to disambiguate these in this study or the future.

[Editors' note: further revisions were requested after re-review, as described below.]

Thank you for resubmitting your work entitled "Conformational change within the extracellular domain of B cell receptor in B cell activation upon antigen binding" for further consideration at eLife. Your revised article has been favorably evaluated by Arup Chakraborty (Senior Editor), a Reviewing Editor, and three reviewers.

The manuscript has been improved but there are some remaining issues that need to be addressed before acceptance, as outlined below:

Summary:

There is a general agreement that the FRET measurements have been improved by the revision and that conformational changes in the extracellular domain are being detected. However, all reviewers felt that the interpretation that these conformational changes are causal for BCR triggering is not demonstrated. In fact, one possibility that was raised is that the conformational change is only observed in the receptor that lacks the cytoplasmic tail of IgG, which is known to have signaling activity. Thus, one possibility not explored by you is that the conformational change results in some mode of negative feedback that is averted by signaling through the cytoplasmic tail of IgG isotype, but not IgM.

Essential revisions:

1) Doubts remain on the physiological meanings of the observed effects, especially with regard to the initiation of BCR signaling. The main conclusion of the authors is that intramolecular conformational changes in surface Ig molecules transport the information of extracellular antigen binding across the plasma membrane to induce intracellular BCR signaling (see subsection “Conformational changes were distinct in soluble IgM and IgM-BCR” and Discussion section). This main conclusion is not justified by the data presented. FRET experiments shown in Figure 3 indicate that those BCR classes with enhanced signaling potency (i.e. those with an Ig-γ tail) did NOT show any intramolecular conformational changes. Hence, if the observed conformational changes are a physiologically meaningful mechanism, it is not to "promote" or "facilitate the intracellular signaling in B cells" as suggested by the authors. In fact, it is well established that surface IgG has a greatly improved signaling capacity compared to IgM because the latter lacks a cytoplasmic signaling tail (Martin and Goodnow, 2002; Wakabayashi et al., 2002; Engels et al., 2009; Liu et al., 2010). These reports are not discussed in the present manuscript. If anything, the presented results could be interpreted as an inhibitory mechanism since the changes are specific to BCRs with reduced signaling capability (i.e. BCRs without an Ig-γ tail). Also, the data shown in Figure 2J are not really supporting the idea that conformational changes in the extracellular domains of IgM drive intracellular signaling, since it is not clear how the 'immunological synapses' of cells with high and low FRET signals look like. A reduced FRET signal could simply be an indicator for efficient antigen binding and efficient BCR clustering, which would explain why those cells have enhanced amounts of phospho-CD79.

Another limitation of the study that remains is the lack of a mechanistic explanation to the main observation, which is that both intra- and intermolecular conformational changes in either surface IgM or the IgG-BCR complex depend on the absence or presence of an intracellular Ig-γ tail. How can the transfer of the IgG tail to IgM prevent a conformational change between Fab and Cμ2 domain? An inside out signaling mechanism can be excluded, since this phenomenon is also observed in 293 cells. The illustrations shown in Figure 7 and the Discussion section do not provide any clues and in combination with the unclear situation regarding the physiological meaning of the observed effects, the impact of the entire study on the field remains limited.

2) The interpretation of the changes in FRET between the N-terminus of the Ig and the Cm2 (or Cg2) is mostly with respect to the putative changes induced by antigen that propagate to the intracellular domains. However, this FRET measures distances across the Fc-Fab hinge region, so it is likely that the majority of the movement here involves some stretching or bending at the hinge as two antigens are engaged by the two Fab arms. This should be at least mentioned and included as one interpretation in the manuscript. It is quite possible to imagine that the average location of the Fab arms in IgM vs IgG is different and therefore a different effect may be observed after antigen binding. The effect of the cytosolic tail is potentially very interesting as it suggests that its presence prevents the antigen-induced change. However, in Figure 3, the data show that it is the basal levels of FRET that are changing between constructs, not the antigen-stimulated. Thus, there may be some effect of the intracellular tail on the orientation of the Fab fragments without antigen binding, not necessarily on "conformational changes" induced by antigen. Similar effects of the tail can be seen in FRET with the Igbeta in Figure 6. This again should be included in the authors' interpretation of the data. I think it is a bit premature to conclude that these conformational changes are causal in triggering signaling.

[Editors' note: further revisions were requested prior to acceptance, as described below.]

The manuscript has been improved but there are some remaining issues that need to be addressed before acceptance, as outlined below:

There is a general feeling that you have shown a link between the intracellular composition of the receptor and conformation of the extracellular antigen binding component of the receptor. The mechanism by which this is happening is not clear. We would like to publish your manuscript, but wanted to provide an opportunity for you address some specific comments to present the findings with maximum clarity in light of well known tissue with assessing statistical significance based on p values and related issue of how cells are selected for analysis.

Essential revisions:

1) The major issue is still with the two parts of the paper that asses the effect of the intracellular IgG tail on the FRET in the extracellular domain. Since there is little mechanistic understanding how this can happen, it is important to report on the observations carefully. In Figure 5, based on t-tests, the authors conclude that adding the IgG tail to IgM or removing the tail from IgG changes the FRET levels between the Fc and Ig-β in resting cells. In contrast, in Figure 3, the authors conclude that the effect of the tail on the resting FRET between the Fc and the Fab is not significant and therefore nonexistent. This, however, seem to be just a matter of a p-value cutoff for this particular test, as there is a trend for the FRET changing with p as low as 0.063 in one of the conditions. This creates an odd conclusion – the IgG tail changes neither the resting nor the antigen-induced FRET, yet it changes whether the two are statistically separable from each other. First, it needs to be disclosed if the data in the supplementary figures (Figure 3—figure supplement 1 C,D, or Figure 5—figure supplement 1C,D) are from the same experiments as in the main figures (Figure 3, Figure 5). If yes, the statistical test are not adequate. The whole experiment should be analyzed with ANOVA with corrections for multiple hypothesis testing. Even if the statistical significance is not illuminating, I think it would be good to conclude whether there is a reproducible trend for a change in the FRET levels of the individual conditions or whether simply the data are not strong enough to conclude as to why the tail changes the "response" to antigen.

2) The authors are careful in matching the fluorescent intensity of the cells analysed in the FRET experiments and they do this by selecting cells for the analysis. Presumably there is an increase in BCR fluorescence in cells interacting with antigen. It would be worth explaining in the methods exactly how the cells were selected – are antigen stimulated cells matched to resting or vice versa?

eLife. 2019 Jul 10;8:e42271. doi: 10.7554/eLife.42271.022

Author response

The authors generate some intriguing data that suggest conformational changes of BCR that are induced by antigen on the cell surface, but there is some concern that the authors try to take on too many different questions related to this without establishing a strong enough foundation to analyze the FRET signatures. If the authors were to focus on the site-specific labeling system set up in VRC01 IgM and fully characterize it with all necessary controls, they could have a strong study that may provide an unprecedented picture IgM extracellular domain conformation change on binding of a monovalent antigen.

We appreciate the comments from the editors and the reviewers to our study. As described in detail below, we have tried to address all the questions and comments from the reviewers. Our responses to each individual comment have been integrated into the revised manuscript by adding new data and/or editing the manuscript. We hope our revised manuscript will fulfill the requirements needed for publication in eLife.

Essential revisions:

1) All reviewers were concerned about the use the polyclonal Fab in FRET studies of IgM and IgG. There are 6 Ig domains in IgM Fc and 4 in IgG Fc to which these reagents could bind. So, these reagents were considered inappropriate for the task in terms of both steric effects of Fab multiple binding and the uncertainty as to the location of binding if all sites were not saturated. Unfortunately, none of the experiments using these reagents will be appropriate for publication at the level eLife aspires to. On the other hand, the site specific labeling strategy in the VRCO1 BCR were considered sufficiently site specific and well characterized functionally to be of great use. The authors are urged to build a better foundation around VRC01 binding to gp120 monomers (verify). The reviewers have made some suggestions below regarding possible ways forward that might be achievable in a revised manuscript in 2 months, however, the authors are given latitude to take other approaches that provide interpretable FRET data from a well defined system to provide convincing evidence of conformational changes.

Thank you for all of these insightful suggestions. We agree that for determination of intramolecular FRET between N terminus and Fc region within mIg heavy chain of BCR, site-specific labeling of both positions is necessary. Thus, for measurement of conformational change in the ectodomains of IgM-BCR, we took the suggestion and focused on the VRC01-IgM-BCR with ybbR tag inserted in N terminus and tetracysteine tag inserted in Cμ2 region (Figure 1A). In addition, to compare the conformational change of IgM- and IgG-BCR, we constructed VRC01-IgG-BCR with ybbR inserted in N terminus and tetracysteine tag inserted in Cγ2 region (Figure 1—figure supplement 1) in the revised manuscript. Based on these two VRC01-BCR constructs, we intensely examined the acquired FRET signal of BCR upon binding with HIV-1 gp120 derived-V51 monomer presented on the surface of planar lipid bilayer (Please see Essential revision 3 and 4). Importantly, conformational changes within site-specifically labeled ectodomains of different BCR constructs observed in this revised manuscript are largely consistent with observations generated with polyclonal Fab in NP antigen system in the original manuscript (Figure 2, Figure 3).

For measurement of FRET between Igα/β and mIg heavy chain, we tried to site-specifically label Igβ and mIg heavy chain in IgM-BCR respectively using two strategies: tetracysteine tag for Igβ labeling and ybbR tag for Fc region of mIg labeling, or tetracysteine tag for Fc region of mIg labeling and ybbR tag for Igβ labeling. However, after trying over 10 sites in Fc region of mIg (6 sites in Cμ2, 5 sites in Cμ3) and 4 sites in Igα/β (2 sites in Igα, 2 sites in Igβ), we were still unable to find any positions with ybbR tag insertion can be efficiently labeled by CoA 488 fluorophore (please see representative images Author response image 1). According to the structural study of Igα/β and its assembly with mIg (Radaev et al., 2010), the interaction between Igα/β and Cμ2-Cμ4 of mIg in IgM-BCR might hamper the label of ybbR tag. Considering that FRET between Igβ and mIg in BCR complex results from intermolecular FRET, it is reasonable to use polyclonal Fab fragment of antibody to obtain average FRET (please see Author response image 2) between two distinct neighboring molecules, as several published papers used fluorophore-conjugated polyclonal Fab to measure the intermolecular FRET efficiency, including the intermolecular FRET between neighboring BCR molecules (Tolaret al., 2005; Verveer et al., 2000; Veeriah et al., 2014; Liu et al., 2010). Thus, we used polyclonal Fab for Fc region staining to measure FRET between Igβ and mIg heavy chain in VRC01-BCR, generating data that are consistent with those observations generated with polyclonal Fab in NP antigen system in the original manuscript (Figure 5, Figure 6).

Author response image 2. — Top: FRET between ReAsH and AF647 (binding to IgM Fc5μ, which contains most of Cμ3/4 region) in IgM-BCR. Down: FRET between ReAsH and AF647 in IgG-BCR.

2) The authors should be able to generate CoA488 and ReAsH labeled soluble IgM and study the FRET in solution and the effect of antigen binding with absolute quantification. While this is an IgM antibody, there are ways to produce these in 293 cells without or with J-chain to generate monomers or pentamers. This would enable a systematic study of the conformational dynamics of the receptors in solution with and without ligand and binding and forced clustering. The system could be used to calibrate the FRET signature of cell surface FRET. These experiments could be done in bulk, single cell or microscopy modes.

We took this wonderful suggestion and produced soluble VRC01-IgM monomers (no J-chain) with ybbR tag inserted at N terminus and tetracysteine tag inserted in Cμ2 region in 293F cells, labeling them with CoA 488 and ReAsH (Figure 1—figure supplement 2). Since we used dequencing FRET and FLIM-FRET to examine the FRET efficiency within extracellular domain in VRC01-IgM-BCR, we also took advantage of these two methods for FRET measurement in soluble VRC01-IgM. FRET signals generated with same method in soluble IgM or IgM-BCR are comparable (Figure 2, Figure 2—figure supplement 2), indicating the FRET signal within soluble IgM could be used to calibrate the FRET signal within IgM-BCR. Data generated from both methods showed no conformational change occurred between N terminus and Cμ2 region in soluble IgM upon engagement with V51 monomer antigen presented in lipid bilayer (Figure 2—figure supplement 2). These results are consistent with previous findings (Davies et al., 1983; Davies et al.,1990; Metzger et al.,1978), in which the crystal structures of antibodies indicate that soluble antigen engagement seems incapable of propagating significant conformational changes to membrane-proximal regions of Ig. We also discussed the difference conformational change between soluble IgM and IgM-BCR in the Discussion section.

3) The authors should verify that gp120 is monomeric and could determine the consequences of gp120 binding to the monomeric or pentameric soluble IgM in terms of any evidence for generating of higher order structures, which would suggest that the gp120 is not monomeric or can be induced to form oligomers when engaged by the Fab. But the resulting multimers could be characterized by gel filtration or analytical ultracentrifugation.

We agree with the reviewer on the importance of verifying that gp120 antigen is in monomeric format. We used V51 antigen fragment, which is one epitope-focused antigenic domain recognized by VRC01 antibody and VRC01-BCR expressing B cell as reported in our recent published paper in HIV-1 vaccine studies (Wang et al., 2018). We used blue native-polyacrylamide gel electrophoresis (BN-PAGE) to determine the oligomeric states of V51 fragment (Figure 1—figure supplement 1E). We also used V51 monomer (which has only one epitope recognized by VRC01 antibody) and V51 trimer to stimulate the calcium signal of VRC01-IgM-BCR expressing Ramos B cells. In contrast to the calcium influx induced by V51 trimer, V51 monomer is unable to trigger the activation of B cells (Figure 1—figure supplement 1F).

4) Parts of Figure 1, Figure 2Figure 6 and Figure 7 could be used as a core for a revised paper that provides a better-controlled study that focuses on the gp120 system. The authors may have sufficient time to generate results with solid phase gp120, which can be attached to a solid substrate or presented in supported lipid bilayers.

We agree with the reviewers on this point. In this revised manuscript, we repeat all the FRET measurements with gp120 presented in lipid bilayers (Figure 2B), using these data to replace the data generated with NP system in the original manuscript. In brief, to present gp120 antigen in monomeric form, V51 monomer antigen fragments fused with poly-histidine tag were tethered on the surface of Ni²⁺-containing planar lipid bilayer (PLB) membranes as reported in our published paper (Zhang et al., 2013). In addition, Figure 1, Figure 2, Figure 6 and Figure 7 in the original manuscript were revised according the reviewers’ suggestions and the modified figures (Figure 1, Figure 2, Figure 4 and Figure 5 in the revised manuscript) were used as a core. Data generated with NP system were deleted in the revised manuscript.

The revisions should also address:

1) Does the BCR undergo a conformational change detected in the ectodomains upon antigen binding? With soluble IgM they might hope to be able to model the FRET and even estimate average distances.

We measured the conformational change of soluble tagged VRC01-IgM according to the reviewers’ suggestion (please see Essential revision 2). The results showed that no FRET changes occurred upon membrane-bound V51 monomer antigen engagement (Figure 2, Figure 2—figure supplement 2). Since we are focusing on the conformational changes in BCR complex, it is our opinion that the relative distance change indicated by FRET measurement may reflect the conformational states, as the published papers describing the conformational change of TCR or GPCR after binding with ligand without calculating the accurate distance change (Lee et al., 2015; Vafabakhsh et al., 2015; Nuber et al., 2016).

2) Does the conformational change correlate with downstream activation signaling pathways? The experiments with IgGa/b labeling in the gp120 system may shed light on this.

We quantified the phosphorylated Igα (p-Igα) in stimulated A20II1.6 B cells and examined the association of the level of synaptic recruitment of p-Igα with FRET between N terminus and Cμ2 domain of VRC01-IgM-BCR, using phospho-CD79A (Tyr182) antibody, which has been used for quantifying the strength of B cell activation (Cell Signaling Technology, Volkmann et al., 2016) (Figure 2J), and also determined the association of p-Igα and FRET between mIg heavy chain and Igβ of VRC01-IgG-BCR (Figure 5I). In consistent with the results by measuring the phosphorylated Syk in the NP system from the original manuscript, we found inverse correlation between conformational change and the strength of B cell activation in VRC01-IgM-BCR, in which conformational change occurs in the ectodomains upon antigen engagement. Similarly, in VRC01-IgG-BCR, conformational change in the spatial relationship between mIg heavy chain and Igβ was found to be inversely correlated with the strength of B cell activation.

3) The site specific labeling of the BCR is a novel and useful tool. But to interpret the donor-dequenching data, it is important to know the labelling efficiency with the donor and acceptor. The relative stoichiometry of the labels is particularly critical for comparing the different conditions and BCR constructs. FLIM is a good way to determine the level of FRET irrespectively of labelling stoichiometry, and the authors do use it in one experiment, but unfortunately, they do not derive the information about the fraction of donor molecules in FRET and their FRET level. This could be achieved with soluble IgM and lessons applied to understanding what is happening on the cell surface.

We fully agree with the reviewers that the relative stoichiometry of the labels is critical for comparing FRET changes under different conditions (with or without antigen) or in different BCR constructs (IgM- or IgG-BCR). Although it is difficult to determine the accurate labeling efficiency of the donor and acceptor, respectively, in all the experiments, we analyzed and compared the FRET signals in subsets of cells with equal donor intensity and acceptor intensity (Figure 2D, 2I; Figure 5C, 5H; Figure 3—figure supplement 1; Figure 6—figure supplement 1). For FLIM-FRET method, we showed the fraction of donor molecules in FRET and their FRET level in this revised manuscript (Figure 2H; Figure 5G). We used FLIM-FRET to study the FRET within soluble VRC01-IgM, obtaining the comparable FRET level as VRC01-IgM-BCR on the surface of cells (Figure 2—figure supplement 2C-F). By FLIM-FRET measurement, we found that in contrast to the FRET change occurred in VRC01-IgM-BCR expressed on cell surface upon antigen engagement, such FRET change was not observed in soluble VRC01-IgM after binding with V51 monomer antigen (Figure 2—figure supplement 2).

4) Controls for the FRET photobleaching measurements using donor only and acceptor only samples were missing. These are important as there is often some bleedthrough and also some photobleaching-induced photoconversion. Given that the antigen-induced FRET changes are small, these additional corrections may be significant.

We thank the reviewers for raising this point. We used donor only and acceptor only samples (VRC01-IgM-BCR, in which conformational change occurred within ectodomains) for FRET photobleaching measurements. For FRET between N terminus and Cμ2 domain, we found almost no bleedthrough occurred in donor only or acceptor only sample (please see Author response image 3 and Table A).

For FRET between mIg heavy chain and Igβ, we found weak bleedthrough in donor only or acceptor only sample (please see Author response image 4 and Table B). We calculated the “FRET efficiency” resulted from the bleedthrough in donor only and acceptor only samples (VRC01-IgG-BCR, in which conformational change occurred between mIg heavy chain and Igβ), finding that antigen binding caused no changes on “FRET efficiency” in both samples (please see Table B and Author response image 5), indicating the FRET change upon antigen engagement in VRC01-IgG-BCR was mainly resulted from “true” FRET signals in donor + acceptor samples.

Author response image 5. — Left: donor only samples; Right: acceptor only samples.

	Average CoA 488 MFI Pre-photobleach	Average CoA 488 MFI After-photobleach	Average ReAsH MFI Pre-photobleach	Average ReAsH MFI After-photobleach
Donor only samples	146.6	148.7	1.7	0.9
Acceptor only samples	2.9	3	206.5	8.5

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Table A. Bleedthrough measurement of donor only (CoA 488 labeled at N terminus of mIg heavy chain) and acceptor samples (ReAsH labeled at Cμ2 region of mIg heavy chain) of tagged VRC01-IgM-BCR

	Average ReAsH MFI Pre-photobleach	Average ReAsH MFI After-photobleach	Average AF647 MFI Pre-photobleach	Average AF647 MFI After-photobleach
Donor only samples (Ag-)	187.9	204.6	9.5	1.1
Acceptor only samples (Ag-)	16.4	32.1	171.4	11.8
Donor only samples (Ag+)	192.9	208.5	13.7	1.5
Acceptor only samples (Ag+)	17	31	171.4	19

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Table B. Bleedthrough measurement of donor only (ReAsH labeled in Igβ) and acceptor samples (AF647 labeled in Fcγ region) of tagged VRC01-IgG-BCR

5) Even if the authors don't perform the calibration studies with soluble IgM, they need to deconvolve intermolecular and intramolecular FRET. Its felt that soluble monomers and pentamers will provide critical insight into this, but otherwise another method to look separate out these effects is needed.

We fully agree with the reviewers that intermolecular and intramolecular FRET shall be deconvolved. To exclude potential effects from intermolecular FRET, we performed FRET experiments in 293T cells expressing dually tagged VRC01-IgM-BCR or two types of singly tagged VRC01-IgM-BCR. In detail, plasmids carrying dually tagged VRC01-IgM-BCRs were diluted with plasmids carrying untagged BCRs and expressed in 293T cells to generate only intramolecular FRET (Figure 2—figure supplement 1A), while plasmids carrying two types of singly tagged VRC01-IgM-BCRs (ybbR tag and tetracysteine tag are in different BCRs, respectively) were diluted with untagged BCRs and expressed in 293T cells to serve as a negative control presenting potential intermolecular FRET (Figure 2—figure supplement 1B). We found that antigen binding caused significant lower FRET between CoA 488 and ReAsH in cells expressing dually tagged VRC01-IgM-BCR (Figure 2—figure supplement 1C). Meanwhile, FRET signal between CoA 488 and ReAsH in singly tagged VRC01-IgM-BCRs expressing cells was very low, and no FRET change was observed between these two fluorophores upon antigen engagement (Figure 2—figure supplement 1D), suggesting the alteration of FRET between N terminus and Cμ2 domain within IgM heavy chain generated by antigen-BCR interaction was mainly attributed by the increase of intramolecular distance between these two sites, rather than the intermolecular distance changes. For the suggestion that comparing the FRET within soluble IgM monomers or pentamers, we think the intermolecular FRET measurement in soluble IgM pentamers is not well applied to the IgM-BCRs expressed on the cell surface, since the there is a significant difference between the conditions in which the FRET within soluble IgM was measured and those under which we explored the FRET within BCR complex on cell plasma membrane. For example, it is reported that extracellular conformational change-mediated oligomerization of the BCR requires the WTxxST motif in the transmembrane region of mIg (Tolar et al., 2009), which is lacked in soluble Ig, indicating that interaction between BCR complex and cell membrane might be crucial for conformational change within ectodomains of BCR upon antigen engagement. In addition, according to our previous findings, the activation of IgM-BCR is dependent on mechanical forces generated by the interaction between membrane-bound antigen and surface-expressed IgM-BCR (Wan et al., ELife2015). However, those mechanical forces might be absent or different in the interaction between membrane-bound antigen and soluble IgM.

For the measurement of FRET between mIg heavy chain and Igβ, it is difficult to distinguish the FRET signal within the one BCR complex or between two neighboring BCR complexes. However, considering two reasons, we think the conformational change between mIg and Igβ of VRC01-IgG-BCR observed here was mainly attributed to the FRET signal within the individual BCR complex. First, based on the findings by Tolar et al., 2005), FRET between neighboring BCR complexes increased upon antigen engagement, while in our observation, FRET between mIg and Igβ of VRC01-IgG-BCR decreased, indicating the FRET decrease was mainly explained by the distance change between mIg and Igβ in one BCR complex, rather than the distance change between mIg and Igβ in two distinct BCR complexes. Second, according to the findings in TCR (Lee et al., 2015), the contribution of FRET signal between two adjacent TCR complexes to the whole FRET signal is very low.

6) The authors should provide a better structural model for the conformational changes implied by the FRET measurements. It seems that there are limited number of ways to explain the results and authors should be able to provide schematics to systematically explore these, which may clarify experiments needed to disambiguate these in this study or the future.

We took this suggestion and presented a new model for the conformational changes indicated by the FRET changes (Figure 7).

[Editors' note: further revisions were requested after re-review, as described below.]

There is a general agreement that the FRET measurements have been improved by the revision and that conformational changes in the extracellular domain are being detected. However, all reviewers felt that the interpretation that these conformational changes are causal for BCR triggering is not demonstrated. In fact, one possibility that was raised is that the conformational change is only observed in the receptor that lacks the cytoplasmic tail of IgG, which is known to have signaling activity. Thus, one possibility not explored by you is that the conformational change results in some mode of negative feedback that is averted by signaling through the cytoplasmic tail of IgG isotype, but not IgM.

We fully agree with the editors and reviewers that it is premature to conclude that antigen-induced conformational changes are causal for BCR activation based on the observations in this study. Thus, we have modified the language throughout the revised manuscript, avoiding claiming that BCR conformational change “trigger” or “promote” B cell downstream signaling. For the physiological meanings of the observed BCR conformational changes, we have discussed the possible explanations and future study directions in the following replies to each major point.

Essential revisions:

1) Doubts remain on the physiological meanings of the observed effects, especially with regard to the initiation of BCR signaling. The main conclusion of the authors is that intramolecular conformational changes in surface Ig molecules transport the information of extracellular antigen binding across the plasma membrane to induce intracellular BCR signaling (see subsection “Conformational changes were distinct in soluble IgM and IgM-BCR” and Discussion section). This main conclusion is not justified by the data presented. FRET experiments shown in Figure 3 indicate that those BCR classes with enhanced signaling potency (i.e. those with an Ig-γ tail) did NOT show any intramolecular conformational changes. Hence, if the observed conformational changes are a physiologically meaningful mechanism, it is not to "promote" or "facilitate the intracellular signaling in B cells" as suggested by the authors. In fact, it is well established that surface IgG has a greatly improved signaling capacity compared to IgM because the latter lacks a cytoplasmic signaling tail (Martin and Goodnow, 2002; Wakabayashi et al., 2002; Engels et al., 2009; Liu et al., 2010). These reports are not discussed in the present manuscript. If anything, the presented results could be interpreted as an inhibitory mechanism since the changes are specific to BCRs with reduced signaling capability (i.e. BCRs without an Ig-γ tail). Also, the data shown in Figure 2J are not really supporting the idea that conformational changes in the extracellular domains of IgM drive intracellular signaling, since it is not clear how the 'immunological synapses' of cells with high and low FRET signals look like. A reduced FRET signal could simply be an indicator for efficient antigen binding and efficient BCR clustering, which would explain why those cells have enhanced amounts of phospho-CD79.

Another limitation of the study that remains is the lack of a mechanistic explanation to the main observation, which is that both intra- and intermolecular conformational changes in either surface IgM or the IgG-BCR complex depend on the absence or presence of an intracellular Ig-γ tail. How can the transfer of the IgG tail to IgM prevent a conformational change between Fab and Cμ2 domain? An inside out signaling mechanism can be excluded, since this phenomenon is also observed in 293 cells. The illustrations shown in Figure 7 and the Discussion section do not provide any clues and in combination with the unclear situation regarding the physiological meaning of the observed effects, the impact of the entire study on the field remains limited.

We agree with the editors and the reviewers that physiological meanings of conformational changes within BCR observed in this study need more investigations, thus we should not conclude in this manuscript that conformational changes are causal for BCR-induced B cell activation. As mentioned above, we have modified our description throughout the revised manuscript to avoid drawing these types of conclusions. Moreover, we discussed in the revised manuscript that the actual role (activating or inhibitory effect) of the reported BCR conformational changes in B cell activation can be complex depending on the context of the antigens, the concept of which can be reflected from the investigations on conformational changes of other membrane receptors, such as GPCR. Agonist and antagonist can induce distinct conformational changes of GPCR, indicating that different conformational changes within GPCR can trigger different physiological functions (Olofsson et al., 2014; Vafabakhsh et al., 2015). As for one specific comment from the editors and the reviewers, the observation that conformational change within mIg of IgM-BCR (which was found to be positively associated with the B cell activation level in this study) did not occur in the presence of IgG cytoplamsmic tail seems to contradict with the previous findings, which reported the enhancement of IgG cytoplamsmic tail on the antigen-induced B cell activation (Wakabayashi et al., 2002; Liu et al., 2010; Chen et al., 201). We would like to propose the following two possible explanations, both of which have been included into the Discussion section of the revised manuscript:

One possible explanation is that spatial distance change between N terminus and Cμ2 domain of mIg in IgM-BCR could be interpreted as an inhibitory mechanism, as proposed by the reviewers. Antigen-induced conformational change between N terminus and Cμ2 domain might inhibit further BCR activation through an unexplored negative feedback mechanism (Please see Figure A as below in this document). An interesting hypothesis is that distance change between N terminus and Cμ2 domain might affect the BCR-antigen bond lifetime. As accumulation of bond lifetimes of TCR-pMHC is required for T cell signaling according to a published study (Liu et al.,2014), it shall be intriguing to explore the effect of antigen-induced conformational change on BCR-antigen bond lifetime. This effect could be potentially investigated by combining biomembrane force probe (BFP) technology in the literature and BCR site-specific labeling methodology developed in this study. Since BFP is a highly tech-demanding system, we are going to collaborate with professor Jack Wei Chen in Zhejiang University (please refer to the following link: https://person.zju.edu.cn/en/jackweichen) for this investigation.

Another possible explanation is that conformational change in the spatial relationship between N terminus and Cμ2 (or Cγ2) domain is only required for IgM-BCR (or IgG-BCR equipped with IgM cytoplasmic tail) to transmit antigen-binding signal to membrane-proximal domain, inducing an orientation to expose a clustering interface on the Cμ4 (or Cγ3) domain (Tolar et al.,2009), facilitating BCR triggering (Please see Figure B as below in this document). In contrast, this conformational change is probably not required for IgG-BCR (or IgM-BCR equipped with IgG cytoplasmic tail) to transmit the extracellular signal of antigen-binding to induce the potential conformational change near the Cγ3 (or Cμ4) domain. This hypothesis seems to be consistent with the previous findings that IgG cytoplasmic tail lowers the threshold of mechanical force to induce IgG-BCR activation (Wan et al., 2015; Wan et al.,2018). Considering that the mechanical force might be closely related with the conformational change, it is reasonable to speculate that IgG cytoplasmic tail might lower the threshold of B cell activation through bypassing the required conformational change between N terminus and Cμ2 domain within mIg in IgM-BCR. Thus, it will be constructive to investigate the association between mechanical force and conformational change of BCR in the future study. Combing TGT-NP system described in our previous studies (Wan et al., 2015; Wan et al.,2018) and experimental system that has been established in this manuscript, our future studies will investigate the association between mechanical force and the antigen-induced conformational changes within extracellular domain of BCR.

We also agree with the reviewers that the association between antigen-induced conformational changes and B cell downstream signaling observed in Figure 2J and Figure 4I can be explained by several different ways. Due to the limitation of our experimental system, it is difficult to explore the causal relationship between conformational changes and the recruitment of signaling molecules. It is possible that decreased FRET signal could be a consequence of more efficient antigen binding of the cell, thus enhancing the recruitment of phospho-CD79A, as the reviewers have suggested. It is our opinion that the current bulky-based experimental system may not be able to explicitly address this question. We are now trying to establish a single-molecule FRET experimental system to couple the conformational changes and activation level of individual BCR molecules with equal efficient antigen binding on cell membrane. Since it is a highly tech-demanding system, we are collaborating with professor Chunlai Chen in our university (please refer to the following link: http://life.tsinghua.edu.cn/publish/smkx/11529/2018/20180519040537657202236/20180519040537657202236_.html), who is a co-author of this current manuscript, to accomplish this study.

In addition, we agree with the reviewers that mechanistic explanation to the effect of IgG cytoplasmic tail on the antigen-induced conformational changes of BCR remains to be solved. We speculate that through its interaction with cell membrane (positively charged IgG cytoplasmic tail can associate with the negatively charged acidic phospholipids in the inner leaflet of the plasma membrane, Chen et al., 2015), IgG cytoplasmic tail might alter the basal conformational state of BCR complex (in fact, we found that it can affect the distance between mIg and Igα/β in the absence of antigen, as detailed in reply to major issue #2), or alter the mechanical force sensing capability of BCR (Wan et al.,2018), to influence the antigen-induced conformational changes of BCR. We have included these discussions into the revised manuscript. To test our hypothesis, we are now trying to analyze the structure of BCR complex by cryo–electron microscopy. Structure of IgM- and IgG-BCR equipped with different cytoplasmic tails will be compared, to explore whether the cytoplasmic tail will affect the basal conformational state of BCR complex. Since it is a highly tech-demanding system, we are now collaborating with professor Xueming Li from the School of Life Sciences in our university (please refer to the following link: http://life.tsinghua.edu.cn/publish/smkx/11529/2018/20180519041527828778177/20180519041527828778177_.html) to accomplish this study.

Although the physiological meanings of the observed antigen-induced conformational changes of BCR remains to be solved, in this study, by combining site-specific labeling methodology and FRET-based assay to monitor conformational changes in the extracellular domains within BCR complex upon antigen engagement, we tried to establish new experimental platforms and provide some tentative clues for several long standing questions in antigen receptor biology: How the extracellular signal of antigen binding at the variable region of mIg is transduced to the intracellular ITAMs at the cytoplasmic domain of Igα/β within BCR complex? In detail, whether or not the conformational change occurs within the extracellular domains of BCR upon antigen binding? If yes, where is the exact location of such conformational change; is it at the region within the heavy chain of BCR mIg or between mIg and Igα/β? Whether or not the conformational change within mIg of BCR complex also occurs in soluble Ig molecule upon antigen binding? Whether or not the conformational change of BCR is correlated to the strength of B cell activation? In our opinion, answering these fundamental questions shall be crucial for the further investigation of triggering and regulating mechanisms in BCR activation and B cell function. In this manuscript, conformational changes within mIg and in the spatial relationship between mIg and Igβ were captured and quantified, and the correlation between conformational changes and the strength of BCR activation was also analyzed. Lastly, antigen-binding induced conformational change within mIg of IgM-BCR on cell membrane was not observed in the case of soluble IgM monomer antibodies. It is our opinion that these findings for the first time indicated that antigen-binding induced conformational changes may be truly dependent on the presence of the transmembrane and cytoplasmic domain of BCR and the membrane microenvironment, both of which are not available for soluble antibodies. These results may explain why the published crystal structural studies of antigen-antibody complex indicated that antigen binding does not transmit conformational changes to the membrane-proximal regions of Ig molecule (Metzger, 1974; Metzger, 1992; Davies and Metzger, 1983; Davies et al., 1990). To further compare the structural difference between soluble Ig and BCR on cell membrane, we are now trying to analyze the structure of BCR complex by cryo–electron microscopy with our collaborators as mentioned above.

Thus, all these results in this study may provide molecular explanations for fundamental aspects of BCR activation during transmembrane signaling transduction of B cells. Moreover, experimental systems developed in the present study (i.e. site-specific labeling of BCR and FRET-based assay) also provide a framework from which to examine other ligand-induced molecular events in BCR and other immune receptors.

2) The interpretation of the changes in FRET between the N-terminus of the Ig and the Cm2 (or Cg2) is mostly with respect to the putative changes induced by antigen that propagate to the intracellular domains. However, this FRET measures distances across the Fc-Fab hinge region, so it is likely that the majority of the movement here involves some stretching or bending at the hinge as two antigens are engaged by the two Fab arms. This should be at least mentioned and included as one interpretation in the manuscript. It is quite possible to imagine that the average location of the Fab arms in IgM vs IgG is different and therefore a different effect may be observed after antigen binding. The effect of the cytosolic tail is potentially very interesting as it suggests that its presence prevents the antigen-induced change. However, in Figure 3, the data show that it is the basal levels of FRET that are changing between constructs, not the antigen-stimulated. Thus, there may be some effect of the intracellular tail on the orientation of the Fab fragments without antigen binding, not necessarily on "conformational changes" induced by antigen. Similar effects of the tail can be seen in FRET with the Igbeta in Figure 6. This again should be included in the authors' interpretation of the data. I think it is a bit premature to conclude that these conformational changes are causal in triggering signaling.

Thanks for all of these insightful comments. We fully agree that distance change between N terminus and Cμ2 region within mIg of IgM-BCR observed in this manuscript involves stretching or bending of the hinge region between Fab and Fc fragment. Thus, we have included this interpretation into the Discussion section and Figure legend of Figure 6. In fact, considering that IgD-BCR activation is regulated by its hinge region (Übelhart et al., 2015), it is worth exploring the effect of hinge region on antigen-induced conformational changes of BCR in the future studies.

According to the reviewers’ suggestion, we also investigated the effect of cytoplasmic tail on the basal spatial relationship between Fab and Fc, or between Fc and Igβ within VRC01-BCR without antigen binding. We found that the presence of IgG cytoplasmic tail significantly increased the proximity between Fc and Igβ within VRC01-BCR without antigen binding. In contrast, IgG cytoplasmic tail had no significant influence on the relative distance between N terminus and Cμ2 (or Cγ2) region within VRC01-BCR (Please see Figure 3—figure supplement 1C, D, Figure 5—figure supplement 1C, D in the revised manuscript). Thus, we agree with the reviewers that the intracellular tail might affect the interaction between mIg and Igβ even in the case of antigen free, while the intracellular tail might not influence the location of Fab arms of IgM- and IgG-BCR. We have also included these into Discussion section of the revised manuscript.

[Editors' note: further revisions were requested prior to acceptance, as described below.]

The manuscript has been improved but there are some remaining issues that need to be addressed before acceptance, as outlined below:

There is a general feeling that you have shown a link between the intracellular composition of the receptor and conformation of the extracellular antigen binding component of the receptor. The mechanism by which this is happening is not clear. We would like to publish your manuscript, but wanted to provide an opportunity for you address some specific comments to present the findings with maximum clarity in light of well known tissue with assessing statistical significance based on p values and related issue of how cells are selected for analysis.

We highly appreciate the comments from the editors and the reviewers to our study. We fully agree that maximum clarity and accuracy shall be achieved before the publication of our manuscript. As described in detail below, we have tried to revise our manuscript according to all the insightful suggestions and comments as kindly provided by the editors and the reviewers. We truly hope that our revised manuscript now fulfills the requirements/standards needed for publication in eLife.

Essential revisions:

1) The major issue is still with the two parts of the paper that asses the effect of the intracellular IgG tail on the FRET in the extracellular domain. Since there is little mechanistic understanding how this can happen, it is important to report on the observations carefully. In Figure 5, based on t-tests, the authors conclude that adding the IgG tail to IgM or removing the tail from IgG changes the FRET levels between the Fc and Ig-β in resting cells. In contrast, in Figure 3, the authors conclude that the effect of the tail on the resting FRET between the Fc and the Fab is not significant and therefore nonexistent. This, however, seem to be just a matter of a p-value cutoff for this particular test, as there is a trend for the FRET changing with p as low as 0.063 in one of the conditions. This creates an odd conclusion – the IgG tail changes neither the resting nor the antigen-induced FRET, yet it changes whether the two are statistically separable from each other. First, it needs to be disclosed if the data in the supplementary figures (Figure 3—figure supplement 1 C,D, or Figure 5—figure supplement 1C,D) are from the same experiments as in the main figures (Figure 3, Figure 5). If yes, the statistical test are not adequate. The whole experiment should be analyzed with ANOVA with corrections for multiple hypothesis testing. Even if the statistical significance is not illuminating, I think it would be good to conclude whether there is a reproducible trend for a change in the FRET levels of the individual conditions or whether simply the data are not strong enough to conclude as to why the tail changes the "response" to antigen.

Thanks for these insightful suggestions. We care very much the concerns from the reviewers and would like to fully discuss/clarify these points. “First, it needs to be disclosed if the data in the supplementary figures (Figure 3—figure supplement 1C,D, or Figure 5—figure supplement 1C,D) are from the same experiments as in the main figures (Figure 3, Figure 5)”, for this point, the short answer is no. We took the reviewers’ suggestion and investigated the effect of IgG cytoplasmic tail on the basal spatial relationship between N terminus and Cμ2 (or Cγ2) within mIg, or between mIg and Igβ of VRC01-BCR without antigen binding. When doing this comparison, we performed new experiments (Figure 3—figure supplement 1 C, D, and Figure 5—figure supplement 1C,D) with enlarged sample size for these basal FRET calculations instead of using the original data (Figure 3 and Figure 5) with relatively small sample size. Thus, the data in the original supplementary figures (Figure 3—figure supplement 1C,D, and Figure 5—figure supplement 1C,D) were not from the same experiments as in the main figures (Figure 3 and Figure 5), which would not allow us to directly use ANOVA for statistical analysis. However, it shall be noted that the basal FRET comparison results using the original data from the main figures (Figure 3 and Figure 5) as analyzed by two-tailed t-test (shown in Author response image 8) was similar as the results using the new data from the supplementary figures (Figure 3—figure supplement 1C,D, or Figure 5—figure supplement 1C,D) which were also analyzed by two-tailed t-test. The conclusion is that IgG cytoplasmic tail significantly increased the basal proximity between mIg and Igβ, while IgG cytoplasmic tail did not significantly (but a reproducible trend can be consistently observed) increase the basal distance between N terminus and Fc within mIg.

Author response image 8. — From left to right: IgM vs. MMG in Figure 3, IgG vs. GGM in Figure 3, IgM vs. MMG in Figure 5, IgG vs. GGM in Figure 5.

We fully agree with the reviewers on the point that “If yes, the statistical test are not adequate. The whole experiment should be analyzed with ANOVA with corrections for multiple hypothesis testing”. Since both basal and antigen-binding FRET data from the same experiments were available for the main figures (Figure 3 and Figure 5), we thus included these basal FRET data that were from the same experiments and matched to the antigen-binding FRET data into the newly revised and performed ANOVA to analyze the effect of Ig cytoplasmic tail on both basal conformational state and antigen-induced conformational change of BCR as suggested by the reviewers. The newly analyzed results using two-way ANOVA showed a significant interaction effect (when an interaction effect is present, the effect of one factor shall be dependent on the other factor) between Ig cytoplasmic tail and antigen engagement on both FRET within mIg (Figure 3—figure supplement 2) and FRET between mIg and Igβ (Figure 5—figure supplement 2). These results of two-way ANOVA indicated that Ig cytoplasmic tail significantly affected the antigen-induced conformational change within mIg, or conformational change between mIg and Igβ of BCR. In addition, by using two-way ANOVA with Sidak’s correction for multiple comparisons, we found that the presence of IgG cytoplasmic tail significantly increased the basal proximity between mIg and Igβ of BCR (Figure 5—figure supplement 2A, B), while IgG cytoplasmic tail slightly, but non-significantly increased the basal distance between N terminus and Cμ2 (or Cγ2) within mIg of BCR (Figure 3—figure supplement 2A, B). Thus, it is our opinion that results of two-way ANOVA were consistent with results of two-tailed t-test. We have included all these results and thoughts into the revised manuscript.

While the basal FRET level within mIg in BCRs with distinct Ig cytoplasmic tail showed the absence of significant difference, we consistently observed a reproducible trend that IgG cytoplasmic tail slightly increased the basal distance between N terminus and Cμ2 (or Cγ2). We included this into the revised manuscript. It is our own opinion that Ig cytoplasmic tail may affect the antigen-induced FRET change between Fab and Fc region, rather than influence the basal FRET or the FRET upon antigen-binding, although the mechanism is still to be resolved. As discussed in the revised manuscript, it will be constructive to analyze the structure of BCR complex in the future study to find out whether or not IgG cytoplasmic tail can influence the basal conformational state of mIg. It is also worthy to investigate the association between mechanical force and conformational change of BCR, since IgG tail affects the threshold of mechanical force to induce BCR activation (Wan et al., 2015; Wan et al.,2018).

2) The authors are careful in matching the fluorescent intensity of the cells analysed in the FRET experiments and they do this by selecting cells for the analysis. Presumably there is an increase in BCR fluorescence in cells interacting with antigen. It would be worth explaining in the methods exactly how the cells were selected – are antigen stimulated cells matched to resting or vice versa?

We used several strategies to match the fluorescent intensity of cells in control group and antigen group. When capturing images of resting cells, we selected cells with high fluorescent intensity (i.e. in this step, we need to match resting cells to antigen-stimulated cells). In some cases, fluorescent intensity (donor and acceptor) of acquired cells were comparable in control group and antigen group. In other cases, the fluorescent intensity (donor or acceptor) in control group was still lower than antigen group. Thus, we excluded resting cells with relatively low fluorescent intensity and antigen-stimulated cells with relatively high fluorescent intensity to select a subset of cells with comparable fluorescent intensity for further analysis. Please see Author response image 9 for detailed information (an example of matching process for Figure 2I). We also included these in the Materials and methods section of the revised manuscript. It shall be noted that we are also concerned on this selection step and tried to validate if this matching process of fluorescent intensity can affect the result of FRET comparison. First, Author response image 9 indicated that matching process did not change the result of FRET comparison. Second, besides FRET measurement based on acceptor photobleaching method, which might be influenced by the fluorescent intensity of cells, we also confirmed our conclusion by using FLIM-FRET experiment to exclude the impact of the fluorescent intensity on the conclusions (Figure 2E-H, Figure 2—figure supplement 2D-G, Figure 4D-G).

Author response image 9. — Since the fluorescent intensity of donor in control group was significantly lower, a subset of cells with comparable donor fluorescent intensity were selected as shown in red square for further analysis (top). FRET efficiency, mean fluorescent intensity of donor and mean fluorescent intensity of acceptor after matching the fluorescent intensity of control group and antigen group were shown (bottom).

Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

Transparent reporting form

elife-42271-transrepform.docx^{(245.2KB, docx)}

DOI: 10.7554/eLife.42271.019

Data Availability Statement

All data generated or analysed during this study are included in the manuscript and supporting files.

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PERMALINK

Conformational change within the extracellular domain of B cell receptor in B cell activation upon antigen binding

Zhixun Shen

Sichen Liu

Xinxin Li

Zhengpeng Wan

Youxiang Mao

Chunlai Chen

Wanli Liu

Roles

Abstract

Introduction

Results

Site-specific labeling in mIg heavy chain of BCR complex

Figure 1. Site-specific labeling in mIg heavy chain of IgM-BCR.

Figure 1—figure supplement 1. Site-specific labeling in mIg heavy chain of IgG-BCR.

Figure 1—figure supplement 2. Expression of WT and tagged soluble VRC01-IgM monomer.

Conformational change within the mIgM heavy chain of IgM-BCR upon antigen engagement

Figure 2. Conformational change within mIg heavy chain of VRC01-IgM-BCR upon antigen engagement.

Figure 2—figure supplement 1. Conformational change within mIg heavy chain of VRC01-IgM-BCR upon antigen engagement was mainly attributed to the change of intramolecular FRET.

Figure 2—figure supplement 2. Conformational change within Ig heavy chain of soluble VRC01-IgM monomer upon antigen engagement.

Distinguishing intermolecular FRET and intramolecular FRET

Antigen-binding induced conformational change within mIg heavy chain is closely correlated to the strength of B cell activation

Conformational change within mIg heavy chain is specific to membrane IgM-BCR

IgM- and IgG-BCR exhibited distinct conformational change pattern within the mIg heavy chain upon antigen binding

Figure 3. Conformational change within mIg heavy chain is dependent on the cytoplasmic tail.

Figure 3—figure supplement 1. Conformational change within mIg heavy chain is dependent on the cytoplasmic tail.

Figure 3—figure supplement 2. Conformational change within mIg heavy chain is dependent on the cytoplasmic tail.

Site-specific labeling of the extracellular domain of Igβ in BCR complex

IgM- and IgG-BCR exhibited distinct conformational change pattern in the spatial relationship between mIg and Igβ upon antigen binding

Figure 4. Conformational change in the spatial relationship between mIg heavy chain and Igβ in VRC01-IgG-BCR upon antigen engagement.

Figure 4—figure supplement 1. Site-specific labeling in Igβ of IgM-BCR.

Figure 4—figure supplement 2. Site-specific labeling in Igβ of IgG-BCR.

Figure 4—figure supplement 3. Conformational change in the spatial relationship between mIg heavy chain and Igβ in VRC01-IgM-BCR upon antigen engagement.

Figure 5. Conformational change in the spatial relationship between mIg heavy chain and Igβ is dependent on the cytoplasmic tail.

Figure 5—figure supplement 1. Conformational change in the spatial relationship between mIg heavy chain and Igβ is dependent on the cytoplasmic tail.

Figure 5—figure supplement 2. Conformational change in the spatial relationship between mIg heavy chain and Igβ is dependent on the cytoplasmic tail.

Discussion

Figure 6. Cartoon models of BCR conformational change.

Materials and methods

Plasmids, cells, antibodies and reagents

Purification of soluble VRC01-IgM

Site-specific labeling of BCR and soluble Ig

Ca2+ mobilization analysis

V51 monomer binding measurement

Preparation of antigen-containing coverslip

Molecular imaging by TIRFM and confocal fluorescence microscopy

FRET measurement by acceptor photobleaching method

FRET measurement by FLIM

Intracellular immunofluorescence staining and molecular imaging

Acknowledgements

Funding Statement

Contributor Information

Funding Information

Additional information

Competing interests

Author contributions

Additional files

Data availability

References

Decision letter

Roles

Author response

Author response image 1. Labeling of ybbR tag in Fc region or Igα/β of VRC01-IgM-BCR.

Author response image 2. Schematic illustration showing average FRET between ReAsH labeled at Igβ and AF647-conjugated polyclonal Fab fragment of antibody in Fc region.

Author response image 3. Representative images of donor only and acceptor only samples of tagged VRC01-IgM-BCR before and after acceptor photobleaching.

Author response image 4. Representative images of donor only (ReAsH labeled in Igβ) and acceptor only (AF647 labeled in Fcγ region) samples of tagged VRC01-IgG-BCR before and after acceptor photobleaching.

Author response image 5. “FRET efficiency” in donor only (ReAsH labeled in Igβ) and acceptor only (AF647 labeled in Fcγ region) samples of tagged VRC01-IgG-BCR upon antigen engagement.

Author response image 6. Potential inhibitory effect of conformational change between N terminus and Cμ2 domain of mIg in IgM-BCR.

Author response image 7. Potential activating effect of conformational change between N terminus and Cμ2 domain of mIg in IgM-BCR.

Author response image 8. Basal FRET comparison in BCRs with distinct Ig cytoplasmic tail using two-tailed t-test.

Author response image 9. FRET efficiency, mean fluorescent intensity of donor and mean fluorescent intensity of acceptor before matching the fluorescent intensity of control group and antigen group were shown.

Associated Data

Supplementary Materials

Data Availability Statement

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