Skip to main content
Proceedings of the National Academy of Sciences of the United States of America logoLink to Proceedings of the National Academy of Sciences of the United States of America
. 2017 Dec 4;114(51):13543–13548. doi: 10.1073/pnas.1706883115

Structural insights into the roles of the IcmS–IcmW complex in the type IVb secretion system of Legionella pneumophila

Jianpo Xu a,1, Dandan Xu a,1, Muyang Wan a, Li Yin a, Xiaofei Wang a, Lijie Wu b, Yanhua Liu c,d, Xiaoyun Liu c,d, Yan Zhou a,2, Yongqun Zhu a,2
PMCID: PMC5754761  PMID: 29203674

Significance

Type IVb secretion systems are crucial for the pathogenesis of Legionella pneumophila and Coxiella burnetii. IcmS and IcmW are known as adaptor proteins for the Legionella T4BSS and regulate the translocation of many virulent effector proteins into host cells. However, the mechanism by which IcmS–IcmW recognizes its substrates and facilitates their delivery is unclear. We performed structural and biochemical analyses of the IcmS–IcmW complex. We found that the IcmS–IcmW complex harbors a distinct structure and binds its cognate effectors via an extended hydrophobic surface. IcmS–IcmW also functions as an inseparable partner of DotL to assemble a unique type IV coupling protein complex. Our results provide mechanistic insights into the dual roles of the IcmS–IcmW complex in T4BSSs.

Keywords: Legionella pneumophila, type IVb secretion system, type IV coupling complex, adaptor proteins

Abstract

The type IVb secretion system (T4BSS) of Legionella pneumophila is a multiple-component apparatus that delivers ∼300 virulent effector proteins into host cells. The injected effectors modulate host cellular processes to promote bacterial infection and proliferation. IcmS and IcmW are two conserved small, acidic adaptor proteins that form a binary complex to interact with many effectors and facilitate their translocation. IcmS and IcmW can also interact with DotL, an ATPase of the type IV coupling protein complex (T4CP). However, how IcmS–IcmW recognizes effectors, and what the roles of IcmS–IcmW are in T4BSSs are unclear. In this study, we found that IcmS and IcmW form a 1:1 heterodimeric complex to bind effector substrates. Both IcmS and IcmW adopt new structural folds and have no structural similarities with known effector chaperones. IcmS has a compact global structure with an α/β fold, while IcmW adopts a fully α-folded, relatively loose architecture. IcmS stabilizes IcmW by binding to its two C-terminal α-helices. Photocrosslinking assays revealed that the IcmS–IcmW complex binds its cognate effectors via an extended hydrophobic surface, which can also interact with the C terminus of DotL. A crystal structure of the DotL–IcmS–IcmW complex reveals extensive and highly stable interactions between DotL and IcmS–IcmW. Moreover, IcmS–IcmW recruits LvgA to DotL and assembles a unique T4CP. These data suggest that IcmS–IcmW also functions as an inseparable integral component of the DotL–T4CP complex in the bacterial inner membrane. This study provides molecular insights into the dual roles of the IcmS–IcmW complex in T4BSSs.


Type IVb secretion systems (T4BSSs) play key roles in the pathogenesis of many Gram-negative bacterial pathogens, including Legionella pneumophila, the causative agent of a severe form of human pneumonia known as Legionnaires’ disease, and Coxiella burnetii, a zoonotic pathogen that causes human Q fever by injecting a number of virulent effector proteins into host cells to manipulate host signaling (1). The L. pneumophila T4BSS is composed of 27 Dot (defect in organelle trafficking) or Icm (intracellular multiplication) proteins (2, 3), which form two protein subcomplexes in the cell membrane. The first of these subcomplexes is the major core–transmembrane complex, which consists of five proteins, namely, DotC, DotD, DotF, DotG, and DotH (4, 5). DotC, DotD, and DotH form an outer membrane pore, while DotF and DotG are located in the inner membrane and interact with DotC, DotD, and DotH to form a secretion channel (4).

The second subcomplex in the L. pneumophila T4BSS is the type IV coupling protein complex (T4CP), which is located in the bacterial inner membrane and is assembled by DotL, DotM, and DotN (5). DotL has sequence similarities to a family of ATPases including TraG, TrwB, and TraD, which are known as the coupling proteins in type IVa secretion systems (T4ASSs, also known as the VirB/D4 systems) (6). However, DotL contains an additional C-terminal extension region that has ∼200 residues (6). The coupling proteins in T4ASSs function as inner membrane receptors, linking substrates to the secretion machinery and providing energy for substrate translocation via ATPase activity (5).

IcmS and IcmW are small, acidic proteins in L. pneumophila and are known as adaptor proteins for T4BSSs (79). Both IcmS and IcmW are conserved in the T4BSS-containing pathogens C. burnetii and Rickettsiella gryll. It has been suggested that IcmS and IcmW are located in both the cytoplasm and the inner membrane in L. pneumophila. They interact with one another to form a binary complex. It has been reported that the IcmS–IcmW complex can function as type III secretion chaperones (7). This binary complex can bind many important effectors, including WipA, SidJ, SidD, SidF, and SidG, and regulate their secretion. IcmS and IcmW also interact with the C-terminal extension region of DotL and are required for DotL stability during the transition from the exponential to stationary phase of L. pneumophila growth (10, 11). In addition, LvgA, another small, acidic protein in L. pneumophila, can directly interact with IcmS and plays an important role in bacterial intracellular growth in mouse macrophages (12). However, it is unclear how IcmS–IcmW recognizes effector substrates and what the roles of the DotL–IcmS–IcmW and IcmS–LvgA interactions in T4BSSs are. In this study, we performed a biochemical and structural investigation of the IcmS–IcmW complex and revealed the dual roles of IcmS–IcmW in T4BSSs.

Results

IcmS and IcmW Form a 1:1 Heterodimeric Complex.

We confirmed previous observations that show that IcmS and IcmW are localized in both the cytoplasm and the inner membrane in L. pneumophila (8, 11) (Fig. S1A). To investigate how IcmS and IcmW form a binary complex in the cytoplasm, we coexpressed IcmS and IcmW in Escherichia coli and purified the complex to be homogenous. IcmS and IcmW have 114 and 151 amino acids, respectively. The IcmS–IcmW complex rendered an elution peak between 19 kDa and 33 kDa in gel filtration, which suggested that IcmS formed a 1:1 heterodimeric complex with IcmW in solution (Fig. 1A). Coomassie brilliant blue staining of the eluted samples following SDS/PAGE also confirmed that IcmS and IcmW were at an equal molar ratio in the complex (Fig. 1A). IcmS–IcmW has been suggested to bind effector substrates at multiple internal regions (7). To investigate whether IcmS–IcmW binds to effector substrates as a 1:1 heterodimer, we coexpressed the IcmS–IcmW complex with different binding fragments of its effector substrates, including SidF (residues 1–760), SidG (residues 300–600), SidJ (residues 89–265), and SidJ (residues 596–703). Affinity purification revealed that the IcmS–IcmW complex was indeed bound to these effectors as a 1:1 heterodimer (Fig. 1B).

Fig. 1.

Fig. 1.

IcmS and IcmW form a 1:1 heterodimeric complex. (A) IcmS and IcmW form a 1:1 heterodimer in gel filtration. IcmS and IcmW were copurified using a HiLoad Superdex 75 16/600 column. The samples in the elution peak were analyzed via SDS/PAGE with Coomassie blue staining. (B) IcmS and IcmW functions as a 1:1 heterodimer to bind effector substrates. SidF (residues 1–760), SidG (residues 300–600), SidJ (residues 89–265), and SidJ (residues 596–703) were coexpressed with IcmS and IcmW. After GST copurification using glutathione resins, the samples were analyzed via SDS/PAGE with Coomassie blue staining.

The inner membrane localization of IcmS and IcmW is likely due to interactions between IcmS–IcmW and the C-terminal extension region of DotL (10). We further tested whether IcmS–IcmW also functions as a 1:1 heterodimer to bind DotL. First, we narrowed down the IcmS–IcmW binding region in DotL. The fragment including residues 595–783 of DotL could interact with IcmS–IcmW. A truncated fragment including residues 661–773 still had the capability to bind IcmS–IcmW. However, further deletion abolished the interactions between IcmS–IcmW and DotL. We copurified IcmS–IcmW with the truncated fragment that includes residues 661–773 of DotL (hereafter referred to as DotLc). Indeed, IcmS–IcmW acted as a 1:1 heterodimer to bind DotLc and formed a 1:1:1 ternary complex in gel filtration (Fig. S1B). Taken together, these data suggest that IcmS and IcmW form a 1:1 heterodimeric complex to interact with effector substrates and the T4CP component DotL.

Overall Structure of IcmS–IcmW in Complex with DotLc.

To investigate the mechanism of the IcmS–IcmW complex in T4BSS, we performed structural studies. We failed to crystallize the IcmS–IcmW dimer alone or its complexes with effector substrates. The DotLc–IcmS–IcmW ternary complex was highly stable and was resistant to trypsin digestion and high concentration of salt in solution (Fig. S1 C and D). We succeeded in crystallizing the ternary complex and determined its structure at a resolution of 2.6 Å (Table S1).

The overall structure of the DotLc–IcmS–IcmW ternary complex adopts an inverted “U” shape (Fig. 2A). IcmS and IcmW are located at the two bottom arms of the complex. DotLc covers the IcmS–IcmW dimer from the upper side. In the complex, the IcmS–IcmW dimer exhibits a dumbbell-shaped architecture (Figs. 2A and 3A). Both IcmS and IcmW adopt new structural folds. They do not show any structural similarities with known proteins. IcmS has a compact global structure with an α/β-fold, which contains five α-helices (α1–5) and two β-strands (β1–2) (Fig. 2B). The three relatively longer helices of IcmS, α1, α3, and α4, stack obliquely. The two short helices, α2 and α5, bind to the three longer helices from the bottom side. The β1 and β2 strands form an antiparallel β-sheet, which stacks with α3 and α4 from the upper side (Fig. 2B). Unlike the α/β architecture of IcmS, IcmW has a fully α-folded structure, which contains eight helices (α1–8) (Fig. 2C). The most notable feature of the IcmW structure is that the last two helices (α7–8) in the C terminus protrude out from its main body and are bound by IcmS. In the IcmS–IcmW–DotLc ternary complex, DotLc contains a long loop at its N terminus, followed by three α-helices (Fig. 2A). This long loop mainly winds around the two N-terminal α-helices (α1 and α2) of IcmW. The two middle α-helices of DotLc interact with IcmS and with the C-terminal–protruding α7 and α8 helices of IcmW. The last α-helix (αc) of DotLc extends via a loop and turns vertically to insert into a large groove between α3 and α4 of IcmW at the arm side of the ternary complex (Fig. 2A).

Fig. 2.

Fig. 2.

Structure of IcmS–IcmW in complex with DotLc. (A) The overall structure of the IcmS–IcmW–DotLc ternary complex. IcmS, IcmW, and DotLc are colored in cyan, green, and red, respectively. The N and C termini of the three proteins are labeled as indicated. (B) The structure of IcmS. The secondary structures of IcmS are labeled as indicated. (C) The structure of IcmW.

Fig. 3.

Fig. 3.

Detailed interactions between IcmS and IcmW. (A) The structure of IcmS–IcmW in the complex with DotLc. (B) Detailed interactions between IcmS and the α7 and α8 helices of IcmW. IcmS, and IcmW are colored in cyan and green, respectively. The interacting residues are shown as sticks. The hydrogen bonds and salt bridges are shown as dashed lines. (C) Mutation effects of the interacting residues in IcmS and IcmW on the IcmS–IcmW interactions. His6–SUMO-IcmS and GST-IcmW were coexpressed in E. coli. The interactions between IcmS and IcmW were examined by Ni-NTA resin affinity copurification.

IcmS Stabilizes IcmW by Binding to Its Protruded C Terminus.

IcmS is largely negatively charged on the surface. The compact structure of IcmS suggests that it is stable in solution. Indeed, IcmS was soluble when expressed alone in E. coli. However, IcmS was prone to aggregation in gel filtration (Fig. S1E). The structure of IcmW adopts a relatively loose architecture (Fig. 2C). The main body of the IcmW structure is formed by helices α1–6 and the N-terminal part of α7. The C terminus of α7 and the whole α8 helix protrude from the main body. The α5 helix is located at the center of the IcmW main body and is surrounded by helices α1–4 and α6. The N-terminal part of α7 binds to a groove between α5 and α6 and interacts extensively with α2, α4, α5, and α6 (Fig. 2C). The extensive interactions of α7 with the main body suggest that α7 plays a key role in IcmW structural assembly.

It has been reported that IcmW protein levels in L. pneumophila are severely reduced in the absence of IcmS (8). The relatively loose structure of IcmW is consistent with the high instability of IcmW. When overexpressed alone in E. coli, IcmW mainly precipitated into inclusion bodies, which indicates that the IcmW protein alone was not well folded. Coexpression with IcmS resulted in high solubility of IcmW (Fig. 1A), which suggests that IcmS stabilizes the IcmW structure and promotes its protein folding. In the complex structure, IcmS interacts with the C-terminal helices α7 and α8 of IcmW (Fig. 3A). The interface includes 20 residues from the α1, α3, and α4 helices of IcmS and from the α7 and α8 helices of IcmW. These interactions are mainly hydrophobic interactions at the center of the interface. Five hydrogen bonds and salt bridges strengthen the IcmS–IcmW interaction at the periphery of the interface (Fig. 3B). We performed double mutations of residues C53 and L87 of IcmS and residues V134 and V143 of IcmW, which are all located at the center of the interface, to serine or aspartic acid residues. We tested the effects of these mutations on the IcmS–IcmW interaction and IcmW stability. The double mutations of IcmS C53S/L87D or IcmW V134D/V143D completely disrupted the IcmS–IcmW interaction and destabilized IcmW, which resulted in the precipitation of IcmW into inclusion bodies during coexpression (Fig. 3C). Therefore, the interactions of IcmS with the protruding C terminus of IcmW are necessary for the structural stability of IcmW.

DotLc Is in an Extended Unfolded Conformation in Complex with IcmS–IcmW.

In the complex with IcmS–IcmW, DotLc adopts an extended unfolded conformation (Fig. S2A). The N-terminal loop and the middle α1 and α2 helices of DotLc assemble in a flat shape and are bound to the upper surface of IcmS–IcmW (Fig. S2B). The C-terminal αc helix of DotLc extends from the flat region and turns vertically to be clamped by the α3 and α4 helices of IcmW (Fig. 2A and Fig. S2C), which results in a horizontal “L” shape of DotLc. The secondary structural elements of DotLc do not have any internal interactions. The IcmS–IcmW complex binds DotLc mainly via hydrophobic interactions. The 31 hydrophobic interacting residues of DotLc constitute a continuous interaction network with IcmS and IcmW (Figs. S2 and S3). There are several hydrogen bonds and salt bridges surrounding these hydrophobic interactions. The large interface between DotLc and the IcmS–IcmW complex has an area of 3,074.5 Å2, which mostly covers the entire upper surface of IcmS–IcmW. Because of this large interface, we generated double or triple mutations of the interacting residues in IcmS, IcmW, or DotLc to test their effects on the interactions between DotLc and IcmS–IcmW. Consistent with our observations of the ternary complex structure, the double mutation of F27 and L128 in IcmW, which both interact with the N-terminal loop of DotLc, abolished the binding of IcmS–IcmW to DotLc (Fig. S3E). The residue L54 of IcmS and L49, V124, and L140 of IcmW interact with the middle and C-terminal α-helices of DotLc. The combined mutations of these residues to alanines also disrupted the IcmS–IcmW interaction with DotLc (Fig. S3E). These mutations had no effect on the stability of the IcmS–IcmW complex. Correspondingly, the DotLc mutants L675A/F698A/L702A, F737A/I751A/L763A, and L675A/L763A could not be bound by IcmS–IcmW during copurification (Fig. S2D). These mutagenesis analyses suggest that these hydrophobic interactions play central roles in the interaction of DotLc with IcmS–IcmW.

The Effector Substrate-Binding Surface of IcmS–IcmW Overlaps with the DotLc-Binding Surface.

To investigate how IcmS–IcmW binds effector substrates, we utilized a UV photocrosslinking assay to identify the effector-binding surface on the IcmS–IcmW complex (Fig. S4A). Based on an orthogonal aminoacyl-tRNA synthetase/tRNA pair, the photocrosslinkable unnatural amino acid p-benzoyl-l-phenylalanine (pBpa) was site-specifically incorporated into IcmS or IcmW at the position encoded by the amber codon (UAG) in E. coli (13). The incorporated pBpa around the effector-binding surface on IcmS or IcmW cross-linked with the effector protein, which was coexpressed with IcmS and IcmW, upon excitation by UV light at a wavelength of 365 nm. The cross-linked sites in IcmS or IcmW were determined by detecting cross-linked products of IcmS or IcmW with the effector protein and were mapped onto the surface of the IcmS–IcmW complex structure.

IcmS–IcmW has many effector substrates and can bind one effector at multiple sites. To map the effector-binding surface as completely as possible, a stable and large effector was needed to serve as the substrate for IcmS–IcmW in photocrosslinking assays. SidF is a large effector substrate of IcmS–IcmW (7). A fragment of SidF including its N-terminal 760 residues (residues 1–760) could interact with IcmS–IcmW and function as its substrate. SidF (1–760) was soluble and stable when expressed alone or when coexpressed with IcmS–IcmW. We coexpressed SidF (1–760) with IcmS–IcmW to map the effector-binding surface. In total, we mutated the codons of 62 surface residues in the IcmS–IcmW complex, including 45 residues from IcmW and 17 residues from IcmS, to TAG codons for pBpa incorporation (Table S2). These residues are distributed on the entire surface of the IcmS–IcmW complex. Detection of the IcmW–SidF and IcmS–SidF cross-linked products by SDS/PAGE revealed that 5 sites from IcmS and 12 sites from IcmW cross-linked with SidF (1–760) upon UV excitation (Fig. 4 and Table S2). Among these sites, E66, K120, F27, V31, and R127 of IcmW and L54 of IcmS had the strongest cross-linking abilities with SidF. We mapped all 17 cross-linkable sites onto the IcmS–IcmW structure. Other than S98 of IcmW, which is located on the arm side of the IcmS–IcmW complex and showed an extremely weak cross-linking ability with SidF, the other 16 sites are all located at the edge of the DotLc-binding surface (Fig. 4D), which indicates an overlapping binding surface between the effectors and DotLc on IcmS–IcmW. Consistently, SidF could not form a tetramer with DotLc during coexpression with IcmS–IcmW. We also used SidD as the substrate of IcmS–IcmW in the photocrosslinking assays. The SidD-binding surface on IcmS–IcmW is similar to the surface that SidF binds to (Figs. S5 and S6). Validation of the DotLc interactions with IcmS–IcmW in photocrosslinking assays further confirmed that the DotLc-binding surface on IcmS–IcmW is the same surface that is observed in the ternary complex structure (Fig. S4 BE). Therefore, IcmS–IcmW binds to effector substrates and DotLc through the same hydrophobic surface.

Fig. 4.

Fig. 4.

Photocrosslinking analysis of the effector-binding surface in IcmS–IcmW. (A and B) Photocrosslinking analysis of the residues in IcmW. The codons of the analyzed residues in IcmW were mutated to TAG for pBpa incorporation during coexpression with IcmS and SidF (residues 1–760). The cross-linking reaction was excited by UV light (365 nm). The cross-linked products of IcmW with SidF were analyzed by SDS/PAGE with anti-HA and anti-Flag antibodies. IcmW in the samples was immunoblotted with the anti-Flag antibody. (C) Photocrosslinking analysis of the residues in IcmS. The cross-linked products of IcmS with SidF (residues 1–760) were analyzed via SDS/PAGE and immunoblotting with anti-HA and anti-Myc antibodies. IcmS in the samples was immunoblotted with the anti-Myc antibody. (D) Mapping the cross-linkable residues on the surface of IcmS–IcmW. The cross-linkable residues of IcmS and IcmW are highlighted in green on the surface of the IcmS–IcmW complex.

IcmS–IcmW Recruits LvgA to DotL and Assembles a Unique T4CP.

It was reported that IcmS could interact with LvgA (12). We examined whether IcmS–IcmW and the DotLc–IcmS–IcmW complex could interact with LvgA. When coexpressed with LvgA in E. coli, IcmS–IcmW indeed interacted with LvgA (Fig. S7A). However, the IcmS–IcmW–LvgA complex could not interact with SidD or SidF substrate (Fig. S7 BD), suggesting that LvgA is not a component of the IcmS–IcmW adaptor complex in the cytoplasm. When coexpressed with the DotLc, IcmS, and IcmW in E. coli, LvgA formed a highly stable tetramer with DotLc, IcmS, and IcmW at an equal molar ratio in gel filtration (Fig. 5A). In contrast, the IcmS–IcmW–LvgA complex could not interact with DotD, an outer membrane component of the L. pneumophila T4BSS (Fig. 5B). We further tested whether the DotLc–IcmS–IcmW–LvgA complex could bind the intracellular component, DotN, or the cytoplasmic domain of the DotM subunit of the T4CP. We found that a longer C-terminal fragment (residue 595–783) of DotL, but not DotLc, could interact with IcmS, IcmW, LvgA, and DotN, and form a stable pentamer in gel filtration (Fig. 5C).

Fig. 5.

Fig. 5.

IcmS and IcmW recruits LvgA to DotL and assembles a unique T4CP. (A) IcmS–IcmW forms a stable tetramer with DotLc and LvgA. The DotLc–IcmS–IcmW–LvgA (residues 1–186) complex was purified using a HiLoad Superdex 75 16/600 column. The samples in the elution peak were analyzed by SDS/PAGE with Coomassie blue staining. (B) IcmS–IcmW and LvgA specifically interacts with DotL, but not DotD. The interaction of DotL or DotD with IcmS–IcmW and LvgA was detected by GST pull-down assay by using GST-fused LvgA as bait after coexpression. (C) Interaction of DotN with the DotL–IcmS–IcmW–LvgA complex. A longer fragment (residues 595–783) of DotL was coexpressed with IcmS–IcmW, LvgA, and DotN in E. coli. The DotN–DotL–IcmS–IcmW–LvgA pentamer was purified using a HiLoad Superdex 200 16/600 column.

The findings that the DotLc–IcmS–IcmW complex was resistant to trypsin digestion and high salt concentrations and that there are extensive hydrophobic interactions between IcmS–IcmW and DotLc suggest that IcmS–IcmW is inseparable from DotL. The identification of the stable DotL–IcmS–IcmW–LvgA tetramer in gel filtration experiments (Fig. 5A) indicates that IcmS–IcmW can not only function as a secretion adaptor in the bacterial cytoplasm, but is also an inseparable, stable binding partner of DotL to recruit LvgA and assemble a unique T4CP in the bacterial inner membrane. This finding is consistent with the previously reported membrane localization of LvgA (12). As expected, the DotLc–IcmS–IcmW–LvgA complex could not bind IcmS/IcmW-dependent effectors, including SidD, SidJ, SidG, and SidF (Fig. S7E). Given that LvgA is also a small acidic protein, similar to type III chaperones, it is possible that the DotLc–IcmS–IcmW–LvgA complex can recruit LvgA-dependent or other effector proteins.

Discussion

T4BSSs play key roles in the pathogenesis of many bacterial pathogens (1). IcmS and IcmW are important adaptor proteins in T4BSSs. In this study, we demonstrated that IcmS and IcmW in the Legionella T4BSS can function as a 1:1 heterodimeric adaptor to bind effector substrates in the bacterial cytoplasm and are also inseparable partners of DotL that recruit LvgA and assemble a unique T4CP in the inner membrane for T4BSSs. IcmS and IcmW have no structural similarities with known export chaperones. DotLc in complex with IcmS–IcmW is in an extended unfolded conformation. The extensive hydrophobic interactions between IcmS–IcmW and DotLc stabilize DotL and prevent its degradation by the bacterial cytoplasmic protease ClpP (5).

Our photocrosslinking assays revealed that the effector substrate-binding surface on IcmS–IcmW overlaps with the DotLc-binding surface, suggesting that IcmS–IcmW utilizes the same binding surface to bind effectors and the T4CP component DotL. Many large effectors in Legionella, including SidJ, SidF, and SidG, have been identified as substrates of IcmS–IcmW (7, 8). These effectors have long sequences of amino acids and contain multiple domains. IcmS–IcmW has multiple binding regions in these effectors. The multiple-binding mode of IcmS–IcmW likely provides an efficient way to maintain the large type IV effector proteins in unfolded conformations and prevent their aggregation during translocation. DotL and effector proteins bind to the same surface on IcmS–IcmW, which indicates that a similar extended unfolded conformation is likely adopted by effectors when they are in complex with IcmS–IcmW. We found that the effector SidJ could not compete with SidF on the purified SidF–IcmS–IcmW complex (Fig. S6), suggesting that an unknown factor is required to release effectors from IcmS–IcmW for effector translocation and adaptor cycling.

Based on our results, we propose a working model for IcmS–IcmW in the T4BSS of L. pneumophila. In the cytoplasm, IcmS and IcmW form a 1:1 heterodimer to interact with effector substrates. The interactions maintain effectors in unfolded or partially unfolded states. Through the help of the ATPase DotB or other unknown factors, these effector substrates are released from IcmS–IcmW and are transferred to the protein coupling complex for T4BSSs. IcmS–IcmW also functions as an inseparable partner of DotL and an integral component of T4CP. IcmS–IcmW stabilizes DotL and recruits LvgA to assemble a unique T4CP with DotL and DotN in the inner membrane. The DotL–IcmS–IcmW–LvgA complex possibly functions as a receptor to recruit LvgA-dependent or other effectors for the Legionella T4BSS. Effector release from the DotL–IcmS–IcmW–LvgA complex is likely carried out by DotL.

Recently, Kwak et al. (14) reported crystal structures of the DotL (residues 590–659)–DotN complex and the DotL (residues 656–783)–IcmS–IcmW–LvgA complex. These two structures highlighted the roles of IcmS, IcmW, and LvgA as inseparable integral components of the DotL–T4CP subcomplex in the inner membrane, which is consistent with our findings in this study. They also observed direct interactions of the DotL–IcmS–IcmW–LvgA complex with two effectors, VpdB and SetA (14). These two effectors share no sequence homology with each other and do not contain classical secretion signals at their C termini for the Dot/Icm T4BSS. There was no evidence that the translocation of VpdB and SetA via the T4BSS was dependent on IcmS or IcmW. These data suggest that VpdB and SetA belong to another group of Leginonella effectors. In our studies, we found that the IcmS–IcmW heterodimeric complex, but not the IcmS–IcmW–LvgA, IcmS–IcmW–DotL, or IcmS–IcmW–DotL–LvgA complexes, could directly interact with and functioned as an adaptor for IcmS/IcmW-dependent effectors, including SidG, SidJ, SidD, and SidF in the cytoplasm. We also found that the effector-binding surface on the IcmS–IcmW complex overlaps with the DotL-binding surface, which suggests that IcmS–IcmW utilizes the same surface to recognize IcmS/IcmW-dependent effectors and interact with the T4CP component DotL. Our studies, together with the discoveries by Kwak et al. (14), reveal the dual roles for the IcmS–IcmW complex in the type IV coupling complex and provide molecular insights into the mechanism of the IcmS–IcmW complex in T4BSS effector secretion.

Materials and Methods

Plasmids, Reagents, Strains, and Protein Purification.

DNA of IcmS, IcmW, LvgA, DotL, DotN, and the effectors SidF, SidD, SidJ, and SidG was amplified from the L. pneumophila Lp02 strain. All recombinant proteins were expressed in the E. coli BL21 (DE3) strain. Plasmids and primers used in this study are listed in Dataset S1. For details, see SI Materials and Methods.

Pull-Down, GST Copurification, and Photocrosslinking Assays.

Ni-NTA resin, anti-Flag beads, or glutathione resin were used for Ni-NTA, Flag pull-down, and GST copurification assays, respectively. The photocrosslinking assay samples were analyzed by SDS/PAGE and immunoblotting. For details, see SI Materials and Methods.

Crystallization and Structure Determination.

The crystal diffraction data of the IcmS–IcmW–DotLc complex were processed with the XDS package (15). The structure was solved in Phenix (16). Model building was carried out in Coot (17). Structure refinement was carried out in Phenix and Refmac5 in CCP4 (18). The final structural model was checked with PROCHECK (19). The data collection and refinement statistics are listed in Table S1. For details, see SI Materials and Methods.

Supplementary Material

Supplementary File
pnas.201706883SI.pdf (1.7MB, pdf)
Supplementary File
pnas.1706883115.sd01.pdf (44.3KB, pdf)

Acknowledgments

We thank Drs. Peng Chen and Zengyi Chang for providing plasmids and reagents for photocrosslinking assays and the staff at beamlines BL17U1 and BL18U1 of the Shanghai Synchrotron Radiation Facility and the National Center for Protein Science Shanghai for assistance with diffraction data collection. This research was supported by National Natural Science Foundation of China Grants 81530068, 81322024, 31370722, 81561130162 (to Y. Zhu) and 81501717 (to Y. Zhou) and the Fundamental Research Funds for the Central Universities. Y. Zhu was awarded the Newton Advanced Fellowship by the Royal Society.

Footnotes

The authors declare no conflict of interest.

This article is a PNAS Direct Submission.

Data deposition: The atomic coordinates and structure factors have been deposited in the Protein Data Bank, www.wwpdb.org (PDB ID code 5XNB).

This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.1073/pnas.1706883115/-/DCSupplemental.

References

  • 1.Nagai H, Kubori T. Type IVB secretion systems of Legionella and other Gram-negative bacteria. Front Microbiol. 2011;2:136. doi: 10.3389/fmicb.2011.00136. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 2.Segal G, Purcell M, Shuman HA. Host cell killing and bacterial conjugation require overlapping sets of genes within a 22-kb region of the Legionella pneumophila genome. Proc Natl Acad Sci USA. 1998;95:1669–1674. doi: 10.1073/pnas.95.4.1669. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 3.Vogel JP, Andrews HL, Wong SK, Isberg RR. Conjugative transfer by the virulence system of Legionella pneumophila. Science. 1998;279:873–876. doi: 10.1126/science.279.5352.873. [DOI] [PubMed] [Google Scholar]
  • 4.Kubori T, et al. Native structure of a type IV secretion system core complex essential for Legionella pathogenesis. Proc Natl Acad Sci USA. 2014;111:11804–11809. doi: 10.1073/pnas.1404506111. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 5.Vincent CD, et al. Identification of the core transmembrane complex of the Legionella Dot/Icm type IV secretion system. Mol Microbiol. 2006;62:1278–1291. doi: 10.1111/j.1365-2958.2006.05446.x. [DOI] [PubMed] [Google Scholar]
  • 6.Buscher BA, et al. The DotL protein, a member of the TraG-coupling protein family, is essential for viability of Legionella pneumophila strain Lp02. J Bacteriol. 2005;187:2927–2938. doi: 10.1128/JB.187.9.2927-2938.2005. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 7.Cambronne ED, Roy CR. The Legionella pneumophila IcmSW complex interacts with multiple Dot/Icm effectors to facilitate type IV translocation. PLoS Pathog. 2007;3:e188. doi: 10.1371/journal.ppat.0030188. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 8.Ninio S, Zuckman-Cholon DM, Cambronne ED, Roy CR. The Legionella IcmS-IcmW protein complex is important for Dot/Icm-mediated protein translocation. Mol Microbiol. 2005;55:912–926. doi: 10.1111/j.1365-2958.2004.04435.x. [DOI] [PubMed] [Google Scholar]
  • 9.Zamboni DS, McGrath S, Rabinovitch M, Roy CR. Coxiella burnetii express type IV secretion system proteins that function similarly to components of the Legionella pneumophila Dot/Icm system. Mol Microbiol. 2003;49:965–976. doi: 10.1046/j.1365-2958.2003.03626.x. [DOI] [PubMed] [Google Scholar]
  • 10.Sutherland MC, Nguyen TL, Tseng V, Vogel JP. The Legionella IcmSW complex directly interacts with DotL to mediate translocation of adaptor-dependent substrates. PLoS Pathog. 2012;8:e1002910. doi: 10.1371/journal.ppat.1002910. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 11.Vincent CD, Friedman JR, Jeong KC, Sutherland MC, Vogel JP. Identification of the DotL coupling protein subcomplex of the Legionella Dot/Icm type IV secretion system. Mol Microbiol. 2012;85:378–391. doi: 10.1111/j.1365-2958.2012.08118.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 12.Vincent CD, Vogel JP. The Legionella pneumophila IcmS-LvgA protein complex is important for Dot/Icm-dependent intracellular growth. Mol Microbiol. 2006;61:596–613. doi: 10.1111/j.1365-2958.2006.05243.x. [DOI] [PubMed] [Google Scholar]
  • 13.Ryu Y, Schultz PG. Efficient incorporation of unnatural amino acids into proteins in Escherichia coli. Nat Methods. 2006;3:263–265. doi: 10.1038/nmeth864. [DOI] [PubMed] [Google Scholar]
  • 14.Kwak MJ, et al. Architecture of the type IV coupling protein complex of Legionella pneumophila. Nat Microbiol. 2017;2:17114. doi: 10.1038/nmicrobiol.2017.114. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 15.Kabsch W. XDS. Acta Crystallogr D Biol Crystallogr. 2010;66:125–132. doi: 10.1107/S0907444909047337. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 16.Adams PD, et al. PHENIX: A comprehensive Python-based system for macromolecular structure solution. Acta Crystallogr D Biol Crystallogr. 2010;66:213–221. doi: 10.1107/S0907444909052925. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 17.Emsley P, Cowtan K. Coot: Model-building tools for molecular graphics. Acta Crystallogr D Biol Crystallogr. 2004;60:2126–2132. doi: 10.1107/S0907444904019158. [DOI] [PubMed] [Google Scholar]
  • 18.Collaborative Computational Project, Number 4 The CCP4 suite: Programs for protein crystallography. Acta Crystallogr D Biol Crystallogr. 1994;50:760–763. doi: 10.1107/S0907444994003112. [DOI] [PubMed] [Google Scholar]
  • 19.Laskowski AR, MacArthur WM, Moss SD, Thornton MJ. PROCHECK: A program to check the stereochemical quality of protein structures. J Appl Crystallogr. 1993;26:283–291. [Google Scholar]
  • 20.Dormán G, Prestwich GD. Benzophenone photophores in biochemistry. Biochemistry. 1994;33:5661–5673. doi: 10.1021/bi00185a001. [DOI] [PubMed] [Google Scholar]
  • 21.Wittelsberger A, Mierke DF, Rosenblatt M. Mapping ligand-receptor interfaces: Approaching the resolution limit of benzophenone-based photoaffinity scanning. Chem Biol Drug Des. 2008;71:380–383. doi: 10.1111/j.1747-0285.2008.00646.x. [DOI] [PMC free article] [PubMed] [Google Scholar]

Associated Data

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

Supplementary Materials

Supplementary File
pnas.201706883SI.pdf (1.7MB, pdf)
Supplementary File
pnas.1706883115.sd01.pdf (44.3KB, pdf)

Articles from Proceedings of the National Academy of Sciences of the United States of America are provided here courtesy of National Academy of Sciences

RESOURCES