Dimerization-dependent Folding Underlies Assembly Control of the Clonotypic αβT Cell Receptor Chains

Background: Assembly of heteromeric membrane protein complexes is monitored prior to their transport to the cell surface.

Results: Each of the αβTCR clonotypic chains comprises one domain that folds upon assembly.

Conclusion: Proper αβTCR biogenesis is scrutinized by assembly-coupled folding steps.

Significance: This study shows how the assembly of heteromeric membrane proteins can be monitored and indicates limitations of the possible T cell repertoire.

Abstract

In eukaryotic cells, secretory pathway proteins must pass stringent quality control checkpoints before exiting the endoplasmic reticulum (ER). Acquisition of native structure is generally considered to be the most important prerequisite for ER exit. However, structurally detailed protein folding studies in the ER are few. Furthermore, aberrant ER quality control decisions are associated with a large and increasing number of human diseases, highlighting the need for more detailed studies on the molecular determinants that result in proteins being either secreted or retained. Here we used the clonotypic αβ chains of the T cell receptor (TCR) as a model to analyze lumenal determinants of ER quality control with a particular emphasis on how proper assembly of oligomeric proteins can be monitored in the ER. A combination of in vitro and in vivo approaches allowed us to provide a detailed model for αβTCR assembly control in the cell. We found that folding of the TCR α chain constant domain Cα is dependent on αβ heterodimerization. Furthermore, our data show that some variable regions associated with either chain can remain incompletely folded until chain pairing occurs. Together, these data argue for template-assisted folding at more than one point in the TCR α/β assembly process, which allows specific recognition of unassembled clonotypic chains by the ER chaperone machinery and, therefore, reliable quality control of this important immune receptor. Additionally, it highlights an unreported possible limitation in the α and β chain combinations that comprise the T cell repertoire.

Introduction

In eukaryotic cells, proteins of the secretory pathway are produced in the endoplasmic reticulum (ER).⁵ In higher eukaryotes, these proteins usually enter the ER co-translationally as unfolded polypeptide chains and must acquire their native structure before traversing further along the secretory pathway. This often includes formation of disulfide bonds and glycosylation as well as subunit assembly, processes that are aided and scrutinized by a comprehensive ER-resident protein folding and quality control (QC) machinery (1, 2). To avoid detrimental effects to the organism, the cell has to distinguish between native proteins, partially folded proteins, and ones that are unable to fold. However, in some cases, this discrimination fails, allowing either the ER exit of potentially harmful molecules or the degradation of proteins that could perform their biological functions (3, 4). Therefore, which features of partially folded or incompletely assembled proteins the ERQC machinery recognizes is a key question. Shortcomings in our understanding are partially due to the limited resolution with which protein folding can be monitored in the cell (5, 6).

To provide molecular insights into processes of ERQC, we focused on the α and β chains of the T cell receptor (TCR). The αβTCR is a longstanding model for the quality control of oligomeric membrane proteins, and a great deal is understood about cellular checkpoints in its assembly (7–15). It is composed of eight polypeptide chains, the clonotypic α and β chains and the invariant co-receptor chains (CD3γ, δ, ϵ, and ζ; Fig. 1A), all of which must assemble correctly for the αβTCR to be transported to the cell surface (16–18). First, the α and β chains pair with their designate CD3 co-receptor subunits, giving rise to α-CD3δϵ and β-CD3γϵ trimers, respectively (19). In each case, complementary basic/acidic residues in the transmembrane segments of these chains guide assembly of the trimers and, as has been dissected in detail for the α chains, provide a means to identify chains that do not assemble with CD3 components (7, 8, 10, 12, 20). Retention motifs in the CD3 subunits appear to play an important role in the final step of αβTCR assembly: interaction of αβ-CD3γδϵ₂ hexamers with ζ₂ homodimers to form the complete αβTCR (21, 22). However, it has remained unclear how assembly of the two α-CD3δϵ and β-CD3γϵ trimers is monitored and what role the various lumenal domains of the clonotypic αβ chains play in ERQC. Combining the individual strengths of in vitro and in vivo experimental approaches, we set out to study the molecular events occurring upon heterodimerization of the TCR α/β chains with a view to deriving general insights into how the ERQC system monitors protein assembly.

FIGURE 1.

Assembly of the TCR α and β chains is a prerequisite for ER exit. A, the αβTCR is composed of two clonotypic chains (α and β, structure on the basis of PDB code 1QSE), the invariable CD3 co-receptor (γδϵ₂), and the ζ dimer (ITAM: immunoreceptor tyrosine-based activation motif). The clonotypic chains comprise one variable (Vα and Vβ, respectively) and one constant domain each (Cα and Cβ, respectively). Intradomain disulfide bonds are shown in yellow. Final dispositions of the various domains and during biosynthesis (in parentheses) are indicated. TM basic (+) and acidic residues (−) are shown. A flexible stalk connects the TM regions with the constant domains. In the complete receptor, the α and β chains are linked via a disulfide bridge in the stalk region. The constructs used in this study are shown in the bottom panel. Cysteines/disulfide bridges and predicted glycosylation sites (NXS/T sequences (X ≠ Pro), gray hexagons) are indicated. B, pulse-chase experiments on the isolated (top panel) or co-expressed (bottom panel) α and β chains devoid of their TM basic residues (α^RK->LL and β^K->L, respectively). Only the Cβ antibody is able to immunoprecipitate the αβ heterodimer (the co-precipitating α chain is marked with a black asterisk). Where indicated, proteins were deglycosylated with Endo H or PNGase F. A red asterisk indicates the Endo H-resistant species. Chase times are shown above the lanes. 2.5 μg of each α chain construct and 1.5 μg of each β chain construct were (co-)transfected, and COS-1 cells were metabolically labeled for 1 h. C, pulse-chase experiments were performed as in B, except that the samples were analyzed under non-reducing conditions. The black arrow indicates the αβ-heterodimer, whereas its Golgi-modified form is marked with a red arrow.

Experimental Procedures

Protein Production and Purification

For in vitro studies, individual constructs were amplified from synthetic TCR genes optimized for Escherichia coli expression (Geneart, Regensburg, Germany) and cloned into the pET28a expression vector (Novagen, Gibbstown, NJ). Expression of the various constructs was performed overnight at 37 °C, and resulted in inclusion bodies. Inclusion bodies were solubilized in 100 mm Tris/HCl (pH 8.0), 10 mm EDTA, 10 mm β-mercaptoethanol, and 8 m urea. Solubilized inclusion bodies were centrifuged (20,000 × g, 30 min, 10 °C). The supernatant was applied to a Q-Sepharose column equilibrated in 100 mm Tris/HCl (pH 8.0), 10 mm EDTA, and 5 m urea. Subsequently, the flowthrough was applied to an SP-Sepharose column equilibrated in the same buffer. None of the proteins of interest bound to either column. Accordingly, the flowthrough of the SP-Sepharose column was collected and dialyzed overnight against 100 mm Tris/HCl (pH 8.0), 3 m guanidinium chloride, 5 mm EDTA, and 1 mm DTT. Subsequently, the proteins were applied to a Superdex 200pg (26/60) gel filtration column (GE Healthcare) equilibrated in the same buffer. Proteins were then diluted to 0.1 mg/ml in 250 mm Tris/HCl (pH 8.0), 250 mm l-arginine, 5 mm EDTA, 0.25 mm GSSG, and 0.25 mm GSH and refolded overnight at 4 °C via dialysis against the same buffer. The refolded proteins were concentrated and applied to a Superdex 200pg (26/60) gel filtration column equilibrated in PBS and, if necessary, additionally to a Superdex200 10/300 GL HPLC column (GE Healthcare) equilibrated in PBS. All plasmids were sequenced, and protein identities were verified by mass spectrometry. For NMR samples, proteins were expressed in M9 minimal medium containing ¹⁵N-labeled ammonium chloride.

Spectroscopic Techniques

All CD spectra were recorded with a Jasco J-710 spectropolarimeter (Jasco, Grossumstadt, Germany) at 25 °C in PBS in 0.2-mm quartz cuvettes at a protein concentration of 35 μm. Spectra were recorded 16 times, averaged, and buffer-corrected. Gradient-selected ¹H-¹⁵N TROSY HSQC spectra of samples in PBS and 90% H₂O/10% D₂O were recorded at 298 K on a Bruker Avance 800 MHz NMR spectrometer with a TCI triple-resonance cryogenic probe with 1024 × 128 complex data points and varying number of scans to account for the different protein concentrations and sensitivity. Spectra were processed with nmrPipe (23).

Assessment of Disulfide Bridge Formation

To monitor disulfide bridge formation between and within different proteins in vitro, the respective proteins were (co-)incubated at 25 °C in PBS supplemented with 1 mm GSH/GSSG each. The concentration of each protein was 35 μm. After 3 h, samples were supplemented with 20 mm N-ethylmaleimide and Laemmli buffer (with or without β-mercaptoethanol) and boiled for 3 min. Proteins were separated on 14% SDS-PAGE gels.

Partial Proteolysis and Mass Spectrometry

Stability against proteolytic digestion was assessed by partial proteolysis experiments. Before the addition of protease, proteins were (co-)incubated for 3 h at 25 °C in PBS at a protein concentration of 35 μm each. If present, GSH and GSSG were used at a concentration of 1 mm each, and any reduction/oxidation reactions were quenched by the addition of 20 mm N-ethylmaleimide prior to addition of the protease. After the preincubation step, trypsin (Trypsin Gold, Promega, Madison, WI) was added at a concentration of 1:80 (w/w). Aliquots were withdrawn after different time points, and the proteolysis was terminated by the addition of Roche complete protease inhibitor without EDTA (Roche Applied Science) and Laemmli buffer (without β-mercaptoethanol) and boiling for 3 min. Proteins were separated on 14% SDS-PAGE gels. Gels were quantified with ImageQuant TL software (GE Healthcare). Mass spectrometry experiments were performed by the St. Jude Proteomics core facility using tryptic digestion combined with LC-MS/MS.

Cell Culture Experiments

Constructs were either amplified from synthetic TCR genes optimized for human expression (A6 TCR) (Geneart) or from synthetic genes with the authentic cDNA sequence of the proteins (HA TCR) and cloned into the pSVL vector. BiP was used in a pMT vector (24). All sequences were verified. Experiments were performed in COS-1 cells that were grown in DMEM supplemented with 10% (v/v) fetal bovine serum, 2 mm l-glutamine, and a 1% (v/v) antibiotic-antimycotic solution (25 μg/ml amphotericin B, 10,000 μg/ml streptomycin, and 10,000 units of penicillin; Cellgro/Mediatech, Manassas, VA) (complete DMEM) at 37 °C and 3% CO₂. COS-1 transfections were carried out for 24 h in p60 dishes using GeneCellin (BioCellChallenge, Toulon, France) according to the protocol of the manufacturer. For metabolic labeling, cells were starved for 30 min in complete DMEM without Met and Cys and subsequently supplemented with 100 μCi/p60 dish of EasyTag^TM EXPRESS35S protein labeling mixture (PerkinElmer Life Sciences), with labeling times indicated in the respective figures. Labeling was carried out in the presence of 10 mm DTT for DTT washout experiments. Prior to lysis, cells were washed twice in ice-cold PBS supplemented with 20 mm N-ethylmaleimide when samples were to be run on non-reducing SDS-PAGE gels. Cells were lysed with radio-immunoprecipitation assay buffer (50 mm Tris/HCl (pH 7.5), 150 mm NaCl, 1.0% Nonidet P40 substitute, 0.5% sodium deoxycholate, 0.1% SDS, 0.1 mm PMSF, and 1× Roche complete protease inhibitor without EDTA) or Nonidet P-40 lysis buffer in the case of chaperone co-immunoprecipitation experiments (50 mm Tris/HCl (pH 7.5), 150 mm NaCl, 0.5% Nonidet P40 substitute, 0.5% sodium deoxycholate, 0.1 mm PMSF, and 1× Roche complete protease inhibitor without EDTA, supplemented with 10 units/ml Apyrase for BiP interaction studies (Sigma-Aldrich, St. Louis, MO)). Immunoprecipitations were performed with antibodies against the TCR Cα domain (TCR1145) or Cβ domain (TCR1151) (Thermo Fisher Scientific, Rockford, IL), the FLAG tag (F7425, Sigma), BiP (25), or the HA tag (produced in our laboratory). 3 μg of antibody or 3 μl of serum were used for each cell lysate of one p60 dish and rotated overnight at 4 °C, and, subsequently, CaptivA^TM PriMAB protein A-agarose (Repligen Bioprocessing, Waltham, MA) was added for 2 h at 4 °C under rotation. Immunoprecipitated proteins were washed three times with radio-immunoprecipitation assay buffer or Nonidet P-40 washing buffer (for chaperone co-immunoprecipitations 50 mm Tris/HCl (pH 7.5), 400 mm NaCl, 0.5% Nonidet P40 substitute, and 0.5% sodium deoxycholate) and eluted with Laemmli buffer for 5 min at 95 °C. For Endo H/PNGase F (New England Biolabs, Ipswich, MA) deglycosylation experiments, proteins were eluted with glycoprotein-denaturing buffer and subsequently treated according to the protocols of the manufacturer. For pulse-chase experiments, cells were washed twice with ice-cold PBS after the labeling step, and then complete DMEM supplemented with additional 2 mm of cold Cys and Met was added for the chase times indicated in the figures.

Results

Heterodimerization of the TCR α and β Chains Is a Prerequisite for ER Exit

To provide insights into the assembly process of the clonotypic TCR α- and β-chains, we used the structurally well characterized human A6 αβTCR (26, 27). Experiments were performed in COS-1 cells, which allow for complete TCR assembly and transport (28). Because this study focused on quality control steps that monitor assembly of the TCR α/β chains, the membrane-integrated basic residues of these chains (Fig. 1A) were mutated to Leu (designated α^RK->LL and β^K->L, respectively). This allows stable membrane integration of the TCR α chain in the absence of CD3 assembly (10, 29) and, therefore, permitted us to focus on the quality control checkpoints for the clonotypic TCR α/β chains downstream of this critical step.

The fate of α^RK->LL and β^K->L was monitored by pulse-chase metabolic labeling experiments in which neither α^RK->LL nor β^K->L acquired Endo H resistance during a 3-h chase, indicative of their retention in the ER (Fig. 1B, top panel). In contrast, when α^RK->LL and β^K->L were co-expressed, α chains could be co-precipitated with anti-Cβ antibodies (Fig. 1B, bottom panel, black asterisk), and an Endo H-resistant, PNGase F-sensitive species was observed after the chase (Fig. 1B, bottom panel, red asterisk), an indication of further sugar modification in the Golgi. When analyzed under non-reducing conditions, the formation of an αβ heterodimer was evident (Fig. 1C, black arrow). This heterodimer became larger in size over time, also suggesting further glycan modification in the Golgi (Fig. 1C, red arrow). Therefore, when their basic transmembrane (TM) residues are mutated and CD3 co-receptor interactions are abolished, the clonotypic TCR α and β chains are retained in the ER when unassembled and can continue to the Golgi upon heterodimerization. This indicates retention motifs that become inactive upon heterodimerization, which are different from the intramembrane basic residues or CD3 association.

The ER Chaperones Calnexin and BiP Recognize Unassembled TCR α and β Chains

As glycoproteins, the TCR α and β chains are substrates of the ER Calnexin (Cnx)/Calreticulin (Crt) lectin chaperone system (30, 31). The action of Cnx, however, seems to be limited to early stages of αβTCR biogenesis (32) and is not essential for the correct assembly of αβTCRs (33). Additionally, the ER Hsp70 orthologue BiP (10, 34) and the ER lumenal large Hsp70 Grp170 (35) bind to TCR α and β chains. The precise role of these chaperones in TCR assembly and their binding sites within the clonotypic TCR chains have remained unclear.

When chaperone interactions were analyzed, we observed stable interaction of Cnx with unassembled α and β chains (Fig. 2A). These interactions were reduced significantly when proteins devoid of their TM regions were analyzed (Fig. 2B). Interaction with the ER-lumenal lectin chaperone Crt was significantly less pronounced and not even detectable for the α chain (Fig. 2, A and B), in agreement with earlier studies (30). The α and β chains can each be structurally divided into two Ig-like ectopic domains (Vα and Cα or Vβ and Cβ, respectively), a flexible stalk that connects the constant domains to the TM regions, the TM regions themselves, and a short cytoplasmic tail (Fig. 2C). To assess the role of these different elements in Cnx interaction and ERQC, we created mutants of α^RK->LL and β^K->L where we individually replaced either the flexible stalk or the cytoplasmic tail with poly-GS sequences of similar length (denoted as α^{RK->LL/stalk->GS} and α^{RK->LL/tail->GS} or β^{K->L/stalk->GS} and β^{K->L/tail->GS}, respectively). None of these mutants became Endo H-resistant over a chase period of 5 h, and all mutants showed similar half-lives (Fig. 2, D and E) and wild-type-like interaction with Cnx (Fig. 2F), arguing that the lumenal domains themselves are key factors in ERQC.

FIGURE 2.

Cnx/Crt interactions and maturation of the TCR α/β chains in vivo. A, to analyze interactions of α^RK->LL and β^K->L with endogenous Crt and Cnx, COS-1 cells were transfected with 4 μg of each TCR construct, metabolically labeled for 16 h, and chased for the indicated times. Lysates were immunoprecipitated with the indicated antibodies, and their association with the lectin chaperones is indicated. m, mock. B, the same as in A, except that constructs devoid of their TM regions (α^ΔTM and β^ΔTM) were used. C, schematic of the αβTCR with the flexible stalks, TM basic residues, and cytoplasmic tails indicated. D, to assess the impact of the flexible stalk and the cytoplasmic tail on ER retention of the TCR α chain, COS-1 cells were transfected with the indicated α chain constructs, metabolically labeled for 1 h, and subsequently either lysed directly or chased for the indicated times. Where indicated, proteins were treated with Endo H. E, experiments similar to those described in D were conducted for the TCR β chain. F, influence of the stalk or the cytoplasmic tail on the interaction of either the TCR α chain or the β chain with Cnx. The antibodies used for immunoprecipitation are indicated below the gel (the anti-Cnx antibody is denoted with Cx). Note that the Cα antibody precipitates an unspecific band the size of Cnx, which is also observed in cells transfected with empty pSVL vector (m, mock). 4 μg of each construct were transfected.

Next we analyzed how the other major chaperone system of the ER, the Hsp70 system centered around BiP (36), recognized the TCR α and β chains. In agreement with data published previously (10, 34, 35) we found both α^RK->LL and β^K->L to be BiP substrates (Fig. 3A). Constructs devoid of their TM regions (α^ΔTM or β^ΔTM, respectively; Fig. 1A) showed interactions with BiP that were comparable with their membrane-integrated counterparts (Fig. 3A), revealing the presence of BiP-binding sites within the ectodomains of both the TCR α and β chains. Conversely, Cnx interactions were reduced significantly when proteins devoid of their TM regions were analyzed (Fig. 2, A and B), indicating that membrane proximity or the TM regions themselves are important for the interaction of TCR α/β chains with the integral membrane protein Cnx (37, 38). To further dissect BiP interactions with the TCR clients, we individually analyzed its interaction with each of the isolated α/β chain domains. We reproducibly observed binding, albeit weak, of BiP to the α chain Cα domain but essentially no binding to Vα (Fig. 3B). The opposite was true in the case of the β chain. The Vβ domain bound to BiP very strongly, whereas we observed very minimal interaction of Cβ with BiP (Fig. 3B). Of note, BiP preferentially bound the less glycosylated form of the β chain (Fig. 3, A and B), whereas Cnx did not show such a preference (Fig. 2A). The absence of glycosylation may facilitate BiP binding by exposing binding sites or because of the absence of competition with lectin chaperone binding (37–39).

FIGURE 3.

Interactions of the TCR α/β chains and domains with BiP in vivo. A, association of BiP with the A6 TCR α and β chains in the presence (α^RK->LL or β^K->L, respectively) or absence of their TM regions (α^ΔTM or β^ΔTM, respectively) was detected by co-immunoprecipitation. 3 μg of the TCR constructs were co-transfected with 2 μg of BiP. Cells were metabolically labeled for 30 min. Chase times and precipitating antibodies are indicated. m, mock transfection. B, BiP binding experiments for the individual A6 TCR α and β chain domains were performed as in A. Variable domains were epitope-tagged as indicated. The two bands observed for the β chain/Cβ domain represent two glycospecies (56).

The Isolated TCR α and β Chains Contain Incompletely Folded Domains

To understand the observed chaperone binding characteristics in more structural detail, we assessed the folding status of the α/β chains and their individual domains in the ER by monitoring intradomain disulfide bond formation in the various α/β chain constructs. To do so, cells were treated with DTT during metabolic labeling to inhibit the formation of disulfide bonds in newly synthesized proteins. Subsequent reculturing in DTT-free media allows oxidation to occur. The presence of a disulfide bond can lead to a more compact state of a protein unfolded in SDS, which may be detected by a modest increase in its mobility on non-reducing SDS-PAGE gels (40). If native disulfide bonds do not form, then it is a clear indication of incomplete folding, whereas, if oxidation occurs, this most often correlates with folding. The α and β chains ran slightly faster on non-reducing SDS-PAGE gels after the removal of DTT (Fig. 4A), indicative of the formation of at least one intradomain disulfide bond. The shift was very small, however, likely because of the large size of the proteins, and it was more readily detectable for constructs devoid of their TM regions (Fig. 4A). When the individual domains were assessed similarly, Vα and both bands of Cβ, which correspond to different glycospecies, showed increased mobility on non-reducing gels after removal of DTT, indicative of oxidative folding. In contrast, Cα had an unaltered mobility, and Vβ actually migrated slightly slower, arguing neither of these domains formed an internal disulfide bond (Fig. 4A). Therefore, in agreement with our BiP binding data, the isolated Cα and Vβ domains appeared to remain incompletely folded.

FIGURE 4.

Redox states of the TCR α/β chains and domains in vivo and chaperone interactions of the HA TCR. A, DTT washout experiments were conducted for the α and β chains in the presence (α^RK->LL or β^K->L, respectively) or absence of their TM regions (α^ΔTM or β^ΔTM, respectively) and their constituent domains. Experiments were performed in COS-1 cells using epitope-tagged constructs (HA or FLAG) to allow immunoprecipitation of all redox species. COS-1 cells were transfected with 4 μg of each TCR construct, metabolically labeled in the presence of 10 mm DTT for 30 min, and subsequently either lysed directly or washed to remove DTT and recultured in complete medium for the indicated times. Immunoprecipitated proteins were analyzed by non-reducing SDS-PAGE. To guide the eye, horizontal dotted red lines are shown at the positions of the reduced species. B, experiments similar to those in A were performed for the anti-HA TCR variable domains. The two bands observed for the HA Vα domain correspond to two glycospecies. C, BiP binding experiments were performed for the anti-HA TCR variable domains. 3 μg of the TCR constructs were co-transfected with 2 μg of BiP. Cells were metabolically labeled for 30 min. Chase times and precipitating antibodies are indicated. m, mock transfection.

Genetic recombination and selection give rise to a different set of variable domains in each TCR, which might affect their folding, whereas Cα and Cβ are conserved between different αβTCRs. We therefore assessed the folding properties of the Vα and Vβ domains derived from another, anti-HA human αβTCR (41). Interestingly, for the anti-HA αβTCR, the Vα domain seemed to be unable to form its disulfide bond, whereas Vβ showed a small shift upon DTT washout, indicative of it forming an intradomain disulfide bond (Fig. 4B). Furthermore, in contrast to the A6 Vα domain, the HA TCR Vα domain strongly bound to BiP (compare Figs. 3B and 4C), whereas binding of the HA Vβ domain was of similar strength as that observed for the A6 Vβ domain (compare Figs. 3B and 4C). Together, this suggests that the folding capabilities of the variable domains from different TCRs vary.

To provide structural insights into the TCR αβ assembly process that are not amenable to in vivo analyses, we performed in vitro studies with purified proteins. To this end, we recombinantly expressed the A6 TCR α and β chains (using soluble constructs devoid of their TM regions, denoted α^ΔTM or β^ΔTM, respectively; Fig. 1A) as well as their individual ectodomains and assessed the folding status by CD and NMR spectroscopy. As expected from the αβTCR crystal structure (Fig. 1A) (26, 27), the far-UV CD spectrum of β^ΔTM displayed typical characteristics of a protein dominated by β sheet secondary structure; namely, a minimum around 220 nm and a maximum around 200 nm (Fig. 5A, blue tracing). However, β^ΔTM displayed low signal intensities around 200 nm, which is indicative of the presence of unstructured regions. This was even more pronounced for α^ΔTM (Fig. 5A, red tracing). To further dissect this finding, we individually analyzed the variable and constant domains. In keeping with our in vivo studies, the far-UV CD spectrum of Vα (Fig. 5B, green tracing) was that of a well folded β sheet protein, whereas Cα showed the spectrum of a predominantly unfolded protein (Fig. 5B, red tracing). We observed a similar behavior for the constituents of the β chain but with the domains switched. Cβ was well folded (Fig. 5B, blue tracing), whereas Vβ was not amenable to structural studies because of its tendency to aggregate. Therefore, it most likely accounts for the less well folded parts within β^ΔTM (compare the spectra in Fig. 5, A and B). In agreement with our CD data, the ¹H-¹⁵N TROSY HSQC NMR spectrum of the Cα domain had limited chemical shift dispersion and narrow line widths, indicating fast interconversion of different conformations. It was therefore typical of a disordered protein lacking a defined structure (Fig. 5C, red spectrum). Of note, the resonances observed for the Cα domain were almost completely superimposable with a subset of signals of the ¹H-¹⁵N TROSY HSQC spectrum obtained for α^ΔTM (Fig. 5C, black spectrum), and the remaining, mostly well dispersed resonances superimposed well with the spectrum of Vα (Fig. 5C, green spectrum). Therefore, also in the context of α^ΔTM, Cα appears to be unstructured, whereas Vα is well folded.

FIGURE 5.

Structural properties of the TCR α/β chains and domains in vitro. A, far-UV CD spectra of α^ΔTM (red) and β^ΔTM (blue). MRW, mean residue weight. B, far-UV CD spectra of Vα (green), Cα (red), and Cβ (blue). Minima around 200 nm are indicative of unstructured regions, whereas β sheet proteins are expected to show a minimum around 220 nm and a maximum around 200 nm. C, ¹H-¹⁵N TROSY HSQC spectra of ¹⁵N-labeled α^ΔTM (black) overlaid with the spectra of ¹⁵N-labeled Vα (green) and ¹⁵N-labeled Cα (red). The inset shows the isolated spectrum of ¹⁵N-labeled α^ΔTM (black). All measurements were performed at 25 °C in PBS, supplemented with 10% D₂O in the case of NMR experiments.

Assembly-dependent Folding of the TCR α and β Chains

Taken together, our in vitro data show that the A6 TCR α and β chains each comprise one well folded domain (Vα and Cβ, respectively) and one less well folded domain in isolation. Cα appeared to be almost completely unfolded both in isolation and within the α^ΔTM chain. In contrast, Vβ seemed to possess some β sheet structure, at least in the context of the complete β^ΔTM chain, as indicated by the presence of more β sheet structure in the CD spectrum of β^ΔTM compared with that of α^ΔTM (Fig. 5A). In vivo, the Vα and Cβ domains of the A6 TCR formed their internal disulfide bridge and only weakly bound to BiP, whereas Cα and Vβ stably bound to BiP and remained reduced (Figs. 3B and 4A). On the basis of this good agreement between our in vitro and in vivo data, we used the recombinant proteins to analyze the assembly process of the α and β chains and the concomitant structural changes in more detail. To this end, we performed partial proteolysis experiments on the recombinant proteins. Products were analyzed by both SDS-PAGE and mass spectrometry. Under non-reducing conditions, α^ΔTM migrated as four distinct bands (Fig. 6A, left panel) that resolved into a single band under reducing conditions (Fig. 6D), indicative of disulfide bonding heterogeneity. Partial proteolysis with trypsin rapidly degraded these four species into a single one that we identified by mass spectrometry to be the Vα domain (Fig. 6A, left panel, and B), arguing that Cα causes the disulfide bonding heterogeneity. Cα contains two cysteines, and the flexible stalk of the α chain contains an additional cysteine that forms an interchain disulfide bond with the β chain in the mature receptor (26, 27) (Fig. 1A). If all redox species of a three cysteine-containing protein are populated, four species are expected, exactly what we observed. In vivo, only a single redox species could be detected (Fig. 4A), arguing that this potential for heterogeneity is suppressed in cells. In comparison to the α chain, the β chain was degraded more slowly by trypsin (Fig. 6A, center panel). All stable fragments contained the Cβ domain (Fig. 6B), in agreement with the finding that Vβ is the less well folded domain. When assembled, the αβ heterodimer only populated a single redox species and was very resistant to proteolytic digestion (Fig. 6, A–C), which indicates assembly-induced folding events in the TCR α and β chains.

FIGURE 6.

Structural changes within the TCR α and β chains upon interaction and identification of minimal interaction elements. A, partial proteolysis of α^ΔTM or β^ΔTM and their covalent heterodimer in vitro. Major proteolytic fragments were analyzed by mass spectrometry (indicated with numbers). Digestion times with trypsin are shown below the gels. Proteins were analyzed by non-reducing SDS-PAGE. B, identified proteins from the proteolytic digest in A. C, quantification of α^ΔTM/β^ΔTM degradation upon trypsin treatment in A. Open symbols show the sum of the band intensities for the isolated α^ΔTM and β^ΔTM chains, and closed symbols show the band intensity of the covalent αβ heterodimer (n = 3, mean ± S.D.). norm., normalized. a.u., arbitrary units. D, disulfide bridge formation was used as a reporter for productive assembly of the different TCR chains and domains. Reactions were carried out in the presence of a GSH/GSSG redox system prior to analysis on non-reducing (left panel) or reducing (right panel) SDS-PAGE gels. Co-incubated proteins are indicated above the lanes. The αβ heterodimer is marked with a black arrow. The most compact α^ΔTM species induced upon co-incubation with β^{ΔTM, ΔCys} is marked with a red arrow. E, left panel, superimposition of the ¹H-¹⁵N TROSY HSQC spectra of the isolated ¹⁵N-labeled α^ΔTM chain (red) with the ¹⁵N-labeled α^ΔTM chain in the presence of unlabeled Cβ (black) demonstrates a lack of structural changes upon addition of Cβ. right panel, superimposition of the ¹H-¹⁵N TROSY HSQC spectra of the isolated ¹⁵N-labeled Cα domain (red) with the ¹⁵N-labeled Cα domain in the presence of β^ΔTM (black) demonstrates the absence of structural changes upon addition of β^ΔTM. Measurements were performed at 25 °C in PBS supplemented with 10% D₂O with a 2-fold excess of the unlabeled proteins over the ¹⁵N-labeled proteins each.

To identify minimal elements needed for αβ heterodimerization and concomitant folding events, we assessed the interaction of the α chain with the Cβ domain and, vice versa, of the β chain with the Cα domain. No covalent dimers could be detected in either case (Fig. 6D), arguing that interaction of the constant domains depends on the additional presence of the variable domains. NMR spectroscopy supported this finding (Fig. 6E). Interestingly, when we used a β chain in which the cysteine residue that forms the interchain disulfide bridge with the α chain was deleted (Fig. 1A, β^{ΔTM, ΔCys}), it was still capable of reshuffling the various α chain redox species and led to the predominant population of a single α chain species in α^ΔTM (Fig. 6D). This strengthens our conclusion that the β chain induces folding of Cα and that assembly-dependent folding does not depend on the presence of the interchain disulfide bond, which is in agreement with the fact that the αβ interchain disulfide bond is dispensable for TCR transport to the cell surface (42).

Taken together, our data suggest a mechanism of reciprocally induced domain folding upon heterodimerization of the TCR α/β chains. Unfolded domains are specifically recognized by ER chaperones, and their folding is a prerequisite for release from the designated chaperones in the ER environment. Together, this allows ERQC to monitor the proper assembly of functional αβTCRs.

Discussion

How comparable data obtained from in vitro analyses are to those from in vivo folding studies is a matter of considerable debate. For all proteins analyzed in this study, a very good agreement existed. For domains that appeared well folded in vitro (A6 Vα and Cβ), we found evidence of disulfide bridge formation and the absence of binding to the Hsp70 chaperone BiP in vivo. Similarly, in vitro patterns signifying unfolded/unstructured domains correlated with stable BiP binding and the absence of disulfide bond formation in vivo (A6 Cα and Vβ). Binding of calnexin was strongly dependent on membrane integration and could therefore not be assessed easily on a single-domain basis. Of note, even though disulfide bond formation can occur in unfolded proteins in vitro, in vivo folding and oxidation seem to be coupled more tightly (24, 43, 44).

The good agreement between our in vitro and in vivo folding data allowed us to use the complementary strengths of these approaches to analyze the structural details underlying ERQC of TCR α/β chain assembly. Our data show that the constant domain of the α chain, Cα, is unfolded and gains structure upon αβ heterodimerization. Before assembling with a β chain, the Cα domain therefore provides interaction sites for ER chaperones, BiP and Cnx, allowing recognition of unassembled α-CD3δϵ trimers. In the case of the β chains analyzed in this study, the Vβ domain is a strong client of BiP, providing a means for retaining β-CD3γϵ trimers. Of note, even though the HA TCR Vβ domain formed its disulfide bond in vivo, it remained a strong BiP substrate. Therefore, BiP binding sites are present even in an oxidized Vβ domain. This implies that, unlike antibodies, where free light chains (LCs) can be secreted whereas unassembled heavy chains (HCs) are retained, both clonotypic chains of αβTCRs provide a focus of ERQC. This difference becomes biologically meaningful when one keeps in mind that antibody LCs do not have any effector functions whereas antibody HCs do, and TCR α-CD3δϵ and β-CD3γϵ trimers might each trigger aberrant signaling when transported to the cell surface (45). ERQC therefore acts on all antibody and TCR components that could be potentially harmful when transported while assembled incompletely. Taken together, only the combined reciprocal folding of both V and C domains upon assembly produces an αβ-CD3γδϵ₂ hexamer that no longer interacts with ER chaperones and can be transported to the Golgi, where ζ₂ homodimers are added to complete the αβTCR. However, each V domain is unique. Our data show that this may cause different folding properties, which indicates some previously unappreciated restrictions on compatible TCR Vα-Vβ pairs and, therefore, the possible T cell repertoire.

The data presented in this study reveal interesting similarities and differences in terms of ERQC mechanisms between the αβTCR and antibodies. For IgG antibodies, the first constant domain (C_H1) of the HC is unfolded while HCs are unpaired and gains structure upon HC-LC assembly. This allows HCs to be retained by BiP in developmentally immature pre-B cells that are not producing LCs and for BiP to be released and antibody secretion to occur in more mature cells that produce LCs (24, 46, 47). Our data indicate, for αβTCRs, that the Cα domain behaves as a similar target of ERQC. Of note, although C_H1 adopts a regular Ig fold when assembled, Cα lacks an entire β strand in its Ig fold and remains flexible even in the αβ heterodimer (26, 27). This might explain why assembly of the TCR constant domains in vitro depends on the additional presence of the variable domains, whereas C_H1 can assemble with its cognate partner domain C_L in vitro in the absence of the variable regions (46). Another distinction is that IgG assembly primarily relies on the BiP-centered Hsp70 system of the ER (48), whereas the lectins Crt and, in particular, Cnx are major chaperones for the αβTCR (30–32, 49). Cα is the most heavily glycosylated of all αβTCR domains (Fig. 1A), which might facilitate Cnx recruitment to this incompletely folded domain and contribute to the relatively weak BiP binding we observed (38, 39).

Interestingly, in the case of antibodies, the unfolded C_H1 domain is found in HCs, which are expressed first during development. In contrast, the incompletely folded Cα domain is found in the TCR α chain, which developmentally corresponds to antibody LCs, and not in the β chain, which is expressed first (50, 51). For both arms of the adaptive immune response, a mechanism exists for transporting a small amount of the chain that is produced first to the surface for signaling. In the case of pre-B cells, this is achieved by the expression of a surrogate light chain (52) that is able to assemble with HCs to relieve them from ER retention (50, 52, 53). Conversely, immature T cells make a pre-TCRα (54). Even though the pre-TCRα lacks a variable region, it is still able to interact with both the Cβ and the Vβ domain (55) to allow surface expression of the β chain. Our data suggest that Cβ is well folded, which might provide a docking site for pre-TCRα, allowing it to induce further folding or shielding of hydrophobic surfaces in Vβ (55) so that BiP is released and the β chain can be expressed on the cell surface of pre-T cells. This provides a rationale for why pre-TCRα needs to interact with both domains to allow the TCR β chain to leave the ER.

Taken together, our study allows us to propose a detailed model for αβTCR assembly control in the cell (Fig. 7). Even though important differences are observed in ERQC mechanisms overseeing the maturation of the clonotypic chains in αβTCRs and antibodies, the functions of both molecules in the immune response demand highly reliable quality control checkpoints prior to transport to the cell surface. To fulfill these requirements, antibodies and TCRs have evolved quality control steps relying on assembly-induced folding. The choice of domains and even on which chains they are found, in terms of the developmental sequence of expression, argues that this critical step in ERQC for immune receptors has arisen twice through convergent evolution.

FIGURE 7.

A model for αβTCR assembly control in the cell. Unassembled clonotypic α and β chains are both Cnx substrates. The α chain comprises either one (Cα) or two (Cα and Vα) incompletely folded domains. The β chain Cβ domain is well folded whereas Vβ, in at least some αβTCRs, is not. All incompletely folded domains provide BiP interaction sites, with particular strong binding to incompletely folded variable domains. Of note, Vβ binds to BiP independently of its capability to form its intradomain disulfide bond. Over time, because of their hydrophilic TM region, unpaired α chains completely enter the ER lumen and are degraded by ER-associated degradation (ERAD) (14, 15, 29, 46). Note that αβ heterodimerization should retain the α chain at the membrane but might fail to integrate its TM region if the α-chain is not assembled with the CD3 complex (7, 8, 10). Correctly assembled α-CD3δϵ and β-CD3γϵ pairs en route to the native receptor, whose interaction with Cnx might be reduced or abolished (32), still provide BiP binding sites in Vβ and, likely, Cα, even though the temporal relationship of Cnx and BiP binding is not entirely clear. Upon αβ heterodimerization, the incompletely folded domains in the clonotypic chains become structured and are therefore released from chaperones. Finally, assembly with ζ dimers in the Golgi allows transport to the cell surface to occur (13, 21, 22, 57).

Author Contributions

M. J. F. and L. M. H. conceived the study. T. M. carried out NMR experiments. All other experiments were performed by M. J. F. and J. B. M. J. F., J. B., T. M., and L. M. H. analyzed the data and wrote the paper.