Structural analysis of ligand-bound states of the Salmonella type III secretion system ATPase InvC.

ARTICLE Structural analysis of ligand-bound states of the Salmonella type III secretion system ATPase InvC Ivonne Bernal 1 | Jonas Römermann 1 | Lara Flacht 1,2 | Michele Lunelli 1 | Charlotte Uetrecht 2,3 | Michael Kolbe 1,4 1 Department of Structural Infection Biology, Center for Structural Systems Biology (CSSB), Helmholtz-Center for Infection Research (HZI), Hamburg, Germany 2 Heinrich Pette Institute, Leibniz Institute for Experimental Virology, Hamburg, Germany 3 European XFEL GmbH, Schenefeld, Germany 4 MIN-Faculty University Hamburg, Hamburg, Germany Correspondence Michael Kolbe, Centre for Structural Systems Biology, Helmholtz-Center for Infection Research, Department for Structural Infection Biology, Notkestraße 85, 22607 Hamburg, Germany. Email: [email protected] Funding information Free and Hanseatic City of Hamburg; German Federal Ministry of Health; Helmholtz Association funding agency IVF (Initiative and Networking Fund); "Promotion of young CSSB scientists" program by the Joachim Herz Stiftung; Leibniz-Gemeinschaft, Grant/Award Number: SAW-2014-HPI-4; European Research Council under the European Community's Seventh Frameweork Programme Abstract Translocation of virulence effector proteins through the type III secretion system (T3SS) is essential for the virulence of many medically relevant Gram-negative bacteria. The T3SS ATPases are conserved components that specifically recognize chaperone–effector complexes and energize effector secretion through the system. It is thought that functional T3SS ATPases assemble into a cylindrical structure maintained by their N-terminal domains. Using size-exclusion chromatography coupled to multi-angle light scattering and native mass spectrometry, we show that in the absence of the N-terminal oligomerization domain the Salmonella T3SS ATPase InvC can form monomers and dimers in solution. We also present for the first time a 2.05 Å resolution crystal structure of InvC lacking the oligomerization domain (InvCΔ79) and map the amino acids suggested for ATPase intersubunit interaction, binding to other T3SS proteins and chaperone–effector recognition. Furthermore, we validate the InvC ATP-binding site by co-crystallization of InvCΔ79 with ATPγS (2.65 Å) and ADP (2.80 Å). Upon ATP-analogue recognition, these structures reveal remodeling of the ATP-binding site and conformational changes of two loops located outside of the catalytic site. Both loops face the central pore of the predicted InvC cylinder and are essential for the function of the T3SS ATPase. Our results present a fine functional and structural correlation of InvC and provide further details of the homo-oligomerization process and ATPdependent conformational changes underlying the T3SS ATPase activity. KEYWORDS ATPase, bacterial pathogenesis, crystallography, multi-angle light scattering, native mass spectrometry, Salmonella enterica, spectroscopy, type III secretion system (T3SS) Significance statement: Pathogenic Gram-negative bacteria use a conserved ATPase to aid the delivery of virulence factors across the type III secretion system (T3SS) during the infection of human cells. Our high-resolution structures of the Salmonella ATPase, named InvC, reveal how ATP-analogues might induce allosteric regulation of its ATPase activity by moving two loops involved in the secretion of virulence factors. These observations shed novel structural and functional insights into the mechanism of activation of T3SS-associated ATPases. Received: 5 June 2019 Revised: 2 August 2019 Accepted: 6 August 2019 DOI: 10.1002/pro.3704 This is an open access article under the terms of the Creative Commons Attribution-NonCommercial License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited and is not used for commercial purposes. © 2019 The Authors. Protein Science published by Wiley Periodicals, Inc. on behalf of The Protein Society. 1888 Protein Science. 2019;28:1888–1901.wileyonlinelibrary.com/journal/pro 1|INTRODUCTION The injectisome type III secretion system (T3SS) is a multiprotein nanomachine essential for the virulence of many pathogenic Gram-negative bacteria, including Salmonella, Shigella,Yersinia, enteropathogenic Escherichia coli,Chlamydia, and Pseudomonas aeruginosa that cause millions of deaths worldwide each year. 1–3 The T3SS forms a syringe-like structure extending from the bacterial cytosol across the bacterial membranes to the target cell to directly inject virulence effector proteins into its cytoplasm. Although the structural components of the T3SSs are highly conserved among bacterial species, the secreted effectors are pathogen-specific. 1,4–6 Most of the effector proteins require the formation of complexes with their T3SS chaperones prior secretion. The chaperones maintain a region of the effectors partially unfolded to facilitate their subsequent secretion through the narrow aperture of the T3SS channel (20 Å). 7–9 The secretion mechanism of the T3SS critically depends on the hierarchical selection and delivery of effectors by the sorting platform complex. 10 The Salmonella pathogenicity island 1 (SPI-1) sorting platform is a dynamic complex that interacts with the cytosolic interface of the membrane-embedded T3SS and forms cytosolic soluble intermediates. 11,12 This complex is constituted by a central ATPase oligomeric cylinder formed by InvC (SctN in the unified nomenclature), which is linked through its negative regulator OrgB (SctL) to the flagellar C-ring orthologue SpaO (SctQ) and the accessory protein OrgA (SctK). Additionally, InvC interacts through its central pore to the stalk protein InvI (SctO). 13,14 InvC has been proposed to recognize and dissemble the chaperone–effector complexes and unfold the effectors in an ATP-dependent manner. 7 This function mirrors the AAA+ ATP-driven translocase mechanism, which consists of hydrolyzing ATP to power conformational changes of its homo-hexameric cylinder architecture and to trigger unfolding and translocation of substrates through the central pore of the cylinder. 15 However, the T3SS ATPases share high sequence homology and three-dimensional structural similarities with the βsubunit of the F 1 F O ATPases. 16,17 Recently, the E. coli ATPase EscN has been shown to form a homo-hexameric cylinder with six ATP-binding sites located at the interface of adjacent dimer pairs, suggesting cooperativity between subunits. 18 EscN binds to its central stalk protein and presents different functional states supporting a rotary catalytic mechanism homologous to the F 1 F O ATPases. in vivo and in vitro studies show that the T3SS ATPases form oligomers in solution and in association with the T3SS. The stoichiometry of the complexes in solution range from dimers to dodecamers depending on the bacterial species. 16,19,20 The T3SS ATPases fold in three domains. The N-terminal domain is considered to be important for stable assembly of higher oligomers; it binds to the ATPase negative regulator (OrgB in Salmonella) and presents lipid affinity. 20–24 The predicted ATPase core is the central and most conserved domain. It contains the phosphate-binding loop (P-loop) with the Walker box A motif (GxGKT/S) characteristic for enzymes with ATP activity. 25 The C-terminal domain has moderate sequence similarity among different species and is the potential recognition site for chaperone–effector complexes. 22 The atomic structures for the Shigella Spa47, E. coli EscN, Salmonella SPI-2 SsaN, and Salmonella Flagellar FliI ATPases share high similarity in conformation and ATP-analogues binding states. 16,17,26,27 Although the T3SS ATPases are conserved, little is known about the structure of the Salmonella Typhimurium SPI-1 ATPase InvC. Here, we present the first structure of the Salmonella T3SS ATPase InvC lacking the first 79 residues (Δ79) in the apo-state and in the presence of ATP-analogues. We show that Salmonella InvCΔ79dimerizesinsolutioninthe absence of its N-terminal domain. Our structural assignments allow mapping of amino acids and further interpretation of previous genetic and biochemical analysis fundamental for understanding the function of T3SS ATPases. Additionally, we characterize the structure of InvCΔ79 in presence of ADP or ATPγS. These structures reveal additional conformational changes of two loops outside of the ATP catalytic site that have been shown to be essential in the overall function of InvC. These structural evidences provide insights into the energizing mechanism of T3SS ATPases. 2|RESULTS 2.1 |InvCΔ79 forms monomers and dimers in solution The Salmonella InvC belongs to the conserved family of T3SS ATPases, sharing between 38 and 57% sequence identity with its orthologues (Figure S1). It was reported that the N-terminal domain (amino acids 1–79) of the T3SS ATPases is involved in membrane anchoring and homo-oligomer stabilization. 21,22 When we recombinantly expressed and purified the full length InvC, the N-terminal domain suffered fast proteolysis (data not shown), suggesting that the N-terminal domain or the linker to this domain are flexible regions. Hence, we removed the first 79 amino acids to generate a construct containing the predicted ATPase and C-terminal domains followed by a Strep-Tag that we termed InvCΔ79. The E. coli T3SS ATPase forms a homo-hexameric cylinder with ATP-binding sites located at the interface of adjacent subunits, 18 similar to the F 1 ATPases. We reasoned that interaction between ATPase subunits might occur also in the absence of its N-terminal region and characterized the BERNAL ET AL.1889 molecular size of InvCΔ79 in solution. Size-exclusion chromatography coupled to multi-angle light scattering (SECMALS) analysis of InvCΔ79 resulted in two elution peaks, a major one assigned as Peak I and a minor one named Peak II. The weight-averaged molecular masses of Peaks I and II corresponded well to a monomer (40 kDa) and a dimer (80 kDa) of InvCΔ79, respectively (Figure 1a, S2). Our results are in line with the E. coli ATPase EscNΔ7 lacking the first seven amino acids that forms dimers in solution. 16 Further analysis of elution Peak I by native mass spectrometry (MS) confirmed the predominance of monomers in this fraction even though some dimers were also detected. In contrast, native MS of the Peak II presented mostly dimers and some monomers. The small amount of trimers and tetramers detected are likely unspecific clusters inherent to this method (Figure 1b, Table S1). Together, these results demonstrate that InvCΔ79 lacking the N-terminal domain exists predominantly as monomer in solution and can self-associate into dimers. 2.2 |Structure of InvCΔ79 To determine the structure of InvC, we performed crystallization trials using the monomeric size-exclusion chromatography (SEC) fraction of InvCΔ79. We solved the X-ray crystal structure of InvCΔ79 in the absence of ATP-analogues at 2.05 Å resolution (Figure 2, Table 1). The apo-form folds in two structural domains, the ATPase core (amino acids R81 to T355) and the C-terminal domain (T356 to N431) similar to its bacterial orthologues. The ATPase catalytic core is constituted by the α/βRossmann fold 25 with a parallel nine-stranded twisted β-sheet flanked by three helices at one side, and four helices at the other one. The phosphate-binding loop motif (P-loop) is constituted by the amino acid sequence GCGKT (162–166) and is located between α2andβ5 of the ATPase core. The smaller C-terminal domain is composed of three helices and contains a helix–loop–helix motif that is proposed to interact with chaperone and effector proteins. 27,28 Analysis of the B-factor values of InvCΔ79 after refinement shows that the ATPase core has an average value of 50.7. Most of this domain is rather rigid (blue) and contains two protruding loops with higher mobility (cyan to red in Figure 3a). The first loop region is located between α5 and α6 (amino acids A255 to L282) and the second is between β9 and α7 (L304 to S320). The average B-factor of the Cterminal domain is 75.3, showing a higher mobility in comparison with the ATPase core. This domain contains a third highly mobile loop region between α10 and α11 (K368 to R400). The β9-α7 loop is highly conserved among the T3SS ATPases and the other two loops show a moderate sequence homology (Figure S1). The three loops are arranged in the same front of InvCΔ79 forming a mobile interface that could potentially be involved in conformational changes or interaction with other molecules during the ATPase activity. Taken together, InvCΔ79 shows high three-dimensional structural similarity with other T3SS ATPases including Spa47 (RMSD of 1.05 Å for 325 aligned Cαresidues), EscN (1.51 Å for 312 residues) (Figure S3), SsaN (1.63 Å for 293 residues), and FliI (1.60 Å for 317 residues). As predicted from the sequence alignment (Figure S1), the major differences between T3SS ATPase structures are located at the C-terminal domains, probably due to its suggested function in recognizing specific effector-bound chaperones. 7,27 2.3 |Structural mapping of functional amino acids Mutational analysis of InvC and its Shigella orthologue Spa47, identified residues R189 and R191 of the ATPase core (marked in lilac in Figure 2) as essential for ATPase activity, homo-oligomerization, and type III secretion. 22,29 In our structure, these amino acids are located at the exposed surface of InvCΔ79 and their side chains present high FIGURE 1 Stoichiometry of InvCΔ79. (a) SEC-MALS analysis of InvCΔ79. The SEC profile (dRI, left axis) presents two elution peaks, I and II. The weight-averaged molar masses (gray, right axis) across the elution peaks correspond to monomeric (40 kDa) and dimeric (80 kDa) states of InvCΔ79. (b) Representative native MS analysis of SEC-peaks I and II demonstrating the monomeric (39,901.9 ± 0.6 Da) and dimeric state (79,808 ± 2 Da) of InvCΔ79, respectively. Dark gray bands highlight corresponding peaks from the two spectra. Masses are summarized in Table S1 1890 BERNAL ET AL. mobility as denoted by their B-factors (Figure 3a). It can be conceived that R189 and R191 play a role in dimerization of InvCΔ79 in the absence of the N-terminal domain. The C-terminal domain of some T3SS ATPases contains conserved amino acids that are crucial for type III secretion. The InvC amino acid Y385 was shown to be essential for secretion of late effectors through the T3SS. The previously suggested mechanism included direct interaction of Y385 with effector–chaperone complexes. 28 We show that Y385 forms a hydrogen bond with the side chain of D394, keeping most of its surface area buried within the structure (Figure 3b inlet). However, we cannot discard the possibility of a structural change around this amino acid upon ATP binding or chaperone–effector interaction. The amino acid E384 was reported to participate in the interaction with the stalk protein in Escherichia coli. 18 E384 is surface exposed in our structure, and as Y385, is located in the α10-α11 mobile loop (Figure 3a). Additionally, the InvC L376 was shown to play a role in recognition of chaperone-bound to effectors. 7,27 Analysis of hydrophobicity distribution of InvCΔ79 shows that L376 is part of a hydrophobic patch together with the amino acids L378, F379, I380, and L382 (Figure 3b). These residues, except for L378, are conserved among T3SS ATPases and might be important candidates for chaperone–effector recognition by nonpolar interactions. 2.4 |Conformational changes associated with ATP-analogue binding to InvCΔ79 in solution To understand the molecular mechanism of ATP recognition of InvC, we monitored the conformational changes of InvCΔ79 upon binding of different ATP-analogues by using Fourier-transform infrared (FTIR) and circular dichroism (CD) spectroscopy. ATPγS, AMP-PNP, or ADP supplemented with equimolar concentrations of magnesium ions were used as ligands. FTIR difference spectroscopy of InvCΔ79 with ATP-analogues showed a decrease of absorbance at 1655 cm −1 , indicating a reduction of α-helical content upon ligand binding (Figure 4a). 30 The signal reduction is more pronounced for the ADPand AMP-PNP-bound forms and moderate for the ATPγS-bound form. CD analysis of InvCΔ79 indicates also a reduction of the α-helical content (208 and 222 nm) in the presence of ADP or AMPPNP, while no major intensity change was detected for the ATPγS-bound form (Figure 4b). Taken together, these results show that InvCΔ79 undergoes conformational changes upon binding to ADP and AMP-PNP in solution, whereas the structural changes upon ATPγS interaction are barely detectable. 2.5 |Crystal structures of InvCΔ79 in the presence of ATP-analogues To further analyze the conformational changes of InvCΔ79 upon ligand binding, we performed co-crystallization and soaking experiments of InvCΔ79 with ADP, ATPγS, or AMP-PNP supplemented with magnesium ions. This allowed us to solve the crystal structures of InvCΔ79 cocrystallized with ADP at 2.80 Å resolution and InvCΔ79 bound to ATPγS by soaking experiments at 2.65 Å resolution. However, we could not assign any ligand density for InvCΔ79 with AMP-PNP and magnesium ions. FIGURE 2 Overall architecture of InvCΔ79. (a, b) Two views of InvCΔ79 produced by 90rotation showing secondary structure elements labeled as in Figure S1. The P-loop region is highlighted in orange and previously studied amino acids are presented as sticks. R189 and R191 are related with intersubunit interaction (lilac). 22 Y385 is essential for full type III secretion 28 and the conserved E384 interacts with the stalk protein in EscN (raspberry). 18 L376 interacts with chaperone–effector complexes (green) 7 BERNAL ET AL.1891 In the InvCΔ79 apo-form, the putative ligand-binding site is occupied by several water molecules (Figure 5). In both co-crystal structures, most of the water molecules were replaced by the ligands. The modeling of ADP bound to InvCΔ79 was challenging because of its discontinuous density, likely due to partial occupancy of the site or flexibility of the ligand. We could model the ligand between two clear densities for the adenine and phosphate groups. However, no density for the ribose group was distinguished (Figure 5, S4). The phosphate groups of the ADP molecule form hydrogen bonds with the InvC P-loop amino acids G164 and T166. The adenine group interacts with a water molecule bound to V411 by hydrogen bonds. In the ATPγS bound structure, a magnesium ion is coordinated by the βand γ-phosphates of the ligand, the side chains of D249 and T166, and one water molecule that forms hydrogen bonds with the α-phosphate of ATPγS. The phosphate groups of the ligand interact with the P-loop of InvCΔ79 forming TABLE 1 Data collection and refinement statistics InvCΔ79 InvCΔ79-ATPγS InvCΔ79-ADP Data collection Wavelength (Å) 1.0332 1.0332 1.0332 Space group P 6 5 P6 5 P6 5 Cell dimensions a, b, c (Å) 106.3, 106.3, 73.5 107.9, 107.9, 73.8 107.4, 107.4, 73.5 Resolution (Å) 100–2.05 100–2.65 100–2.80 (2.10–2.05) (2.71–2.65) (2.87–2.80) R merge 0.102 (1.091) 0.100 (1.596) 0.086 (0.804) R meas 0.109 (1.167) 0.106 (1.694) 0.092 (0.864) CC 1/2 99.6 (66.0) 99.9 (54.1) 99.9 (87.5) I/σ(I) 11.35 (1.98) 16.58 (1.53) 13.17 (1.52) Total reflections 234,745 (16,821) 144,020 (9,272) 80,465 (5,394) Completeness (%) 99.9 (99.2) 99.8 (97.5) 98.3 (95.5) Multiplicity 7.9 (7.8) 10.0 (8.9) 6.8 (6.5) Refinement Reflections used 29,752 14,382 11,813 R work /R free 0.168/0.207 0.186/0.224 0.211/0.257 No. atoms Protein 2,711 2,677 2,623 Ligands 8 57 86 Water 152 33 13 B-factors Protein 56.35 78.85 94.55 Ligands 75.96 105.98 136.29 Water 56.00 71.17 83.29 R.M.S. deviations Bond lengths (Å) 0.003 0.003 0.003 Bond angles () 0.615 0.663 0.644 Ramachandran values Favored (%) 97.14 93.88 92.88 Allowed (%) 2.86 5.25 6.23 Outliers (%) 0.00 0.87 0.89 Rotamer outliers (%) 0.69 6.64 6.43 Clashscore 4.61 8.45 9.42 Statistics for the highest-resolution shell are shown in parentheses. 1892 BERNAL ET AL. hydrogen bonds with the amino acids G164 and T166, and a salt bridge with K165. The adenine group is stabilized by π-πstacking with Y338. The interaction of ligands with InvCΔ79 resembles the ATP-analogue binding observed for orthologue T3SS ATPases (Figure 6). 16,17,22,31 Additionally, single mutations of G164 and K165 in the InvC P-loop result in loss of ATP-hydrolysis function. 22 Our structures validate the relevance of this loop for ATP recognition and allow us to further analyze the properties of the remaining InvC ligand-binding site. FIGURE 3 Structural analysis of InvCΔ79. (a) Structure colored by B-factor values. Color-coding bar shows lower (blue) to higher (red) Bfactor values for rigid to mobile regions, respectively. Flexible loops and amino acids for intersubunit interaction are labeled as in Figure 2. (b) Structure depicted by 180rotation colored by higher (red) to lower (white) hydrophobicity of amino acids. The hydrophobic patch containing L376 is shown as inlet. Y385 buried (forming hydrogen bonds with D394) and E384 exposed in the loop are also depicted in the inlet FIGURE 4 Conformational changes of InvCΔ79 upon ligand binding in solution. (a) FTIR difference spectra of InvCΔ79 bound to ATPγS, ADP, and AMP-PNP in reference to its apo-form. (b) Background-corrected CD spectra of InvCΔ79 in the absence and presence of ATP-analogues. Arrows indicate changes of α-helical content upon nucleotide addition BERNAL ET AL.1893 2.6 |Remodeling of the ATP-binding site upon ligand interaction The phosphate groups of the ATP-analogues interact with the InvCΔ79 P-loop. Upon binding of ATPγS, K165 is displaced toward the β-phosphate of the ligand interacting by a salt bridge. The adenine group of the ATP-analogues binds to a hydrophobic pocket formed by Y338, P410, V411, and M167 in the InvC ligand-binding site. This hydrophobic pocket is in closed conformation in the apo-form and is opened upon interaction with ATP-analogues (Figures 5 and 6a,b). The side chain of M167 is oriented toward V411 creating an open cavity to stabilize the adenine group. In addition, the loop including V411 (α11-α12) is located closer to M167 surrounding and further defining the open hydrophobic pocket (Figure 5). A similar pocket is formed in the ligand-binding site of other T3SS ATPases, although the amino acid composition in this region has little sequence similarity. 16,17,31 A comparison with the nucleotide-binding site of the Shigella Spa47 and E. coli EscN T3SS ATPases shows that the adenine double-ring from the ADP-InvCΔ79 structure is rather displaced from the inner region of the hydrophobic pocket but still captured on its surface (Figure 6). 2.7 |Ligand induced conformational changes in two luminal loops Analyzing the overall structures of the apoand ligand bound states, the α-helical content of InvCΔ79 decreased FIGURE 5 Structural changes of the InvCΔ79 ATP-binding site in the presence of ATP-analogues. Analysis of ligand interaction (left column) and electron density maps with 2Fo-Fc contour at 0.8–1.5σ(right column). P-loop (G162 to T166) interacting with the phosphate groups is colored in light orange and other key amino acids for ligand recognition are colored in cyan. Contacts involved in ligand stabilization are indicated by yellow dashed lines. ADP, ATPγS, and magnesium ion are labeled 1894 BERNAL ET AL. modestly upon recognition of ADP. The ADP-InvCΔ79 structure presents loss of α-helical arrangement mostly in the extremes of α10 and α11 facing the α10-α11 loop. The ATPγS-InvCΔ79 structure presents a scarce decrease of α-helical content in α10 (Figures 7 and 8). The decrease of the α-helical content in the ADP-crystal structure and the partial maintenance of the secondary structure in the ATPγS-InvCΔ79 structure follow the same tendency of the structural changes detected by FTIR and CD spectroscopy analysis. FIGURE 6 Opening of the hydrophobic pocket at the InvCΔ79 ATP-binding site and comparison with T3SS ATPase orthologues. Surface and ribbon representations colored by higher (red) to lower (white) hydrophobicity of amino acids showing (a) the Salmonella InvCΔ79 apo-form, (b) InvCΔ79 in presence of ADP (purple) and ATPγS (lime), (c) the Shigella Spa47Δ83 with ATPγS (PDB ID: 5ZT1) and (d) the E. coli EscNΔ102 with ADP (PDB ID: 2OBM). Ligands and side chains of amino acids stabilizing the ligands are depicted as sticks in the same orientation as in Figure 5 FIGURE 7 Overall structural changes of InvCΔ79 in the presence of ADP. (a) 3D structural alignment of the apoform (blue) with the ADP bound form (purple). Differences in loop conformations are highlighted in rectangles and key amino acids colored as in Figure 2a. The β9-α7 loop moves a distance of approximately 7.1 Å. (b) 90 rotation view showing ADP interaction in the ligandbinding site BERNAL ET AL.1895 The apo-, ADPand ATPγS-InvCΔ79 structures showed conformational differences for two of the most mobile loops (α10-α11 and β9-α7) (Figures 7 and 8). The α10-α11 loop is located at the C-terminal domain of InvC and is part of a helix–loop–helix motif. No density was observed for this loop in co-crystals while the protein backbone was better defined in the apo-form, indicating an increase of its flexibility upon ligand interaction (Figure S5). In our structures, α11 is closely followed by V411 of the ligand-binding site. Upon ligand interaction V411 pulls α11 and gets closer to M167 (Figure 5), possiblyaffectingtheα-helical conformation of α11, α10, and the respective loop. Some amino acids relevant for T3SS ATPase function are located in the α10-α11 loop. Indeed, point mutations in the InvC G383A, E384A, Y385A, and G388A were shown to cause a decrease of secretion of late effectors by the T3SS. 28 Additionally, the amino acidL376thatinteractswith chaperone–effector proteins 7 is also part of this loop. The β9-α7 loop is located next to the P-loop and presents significant conformational changes in the presence of ATP-analogues. Upon binding of ADP and ATPγS, the β9-α7 loop swings away by approximately 7.1 and 8.9 Å, respectively (Figures 7 and 8, S5). Point mutations in this region of InvC, including E306A, E308A, E309A, E310A, and D312A, were reported to affect secretion of late effectors by the T3SS. 28 Similarly, the corresponding β9-α7loopoftheShigella Spa47Δ83 was shown to increase its flexibility upon interaction with ATP-analogues. 31 Interestingly, cryo-EM analysis of the E. coli EscN homo-hexameric cylinder showed differences in the α10-α11 loop conformation for each ligand-bound state and differences in the β9-α7 loop upon ligand recognition. 18 To analyze the location of the loops β9-α7andα10-α11 in a putative hexameric complex, we built a model by superposing the InvCΔ79 apo-form to the EscN asymmetric cylinder (PDB ID: 6NJP) using Pymol (Figure 9). 18 In the model, β9-α7 and α10-α11 are located at the inner pore of the cylinder where the stalk protein (InvI in Salmonella) interacts with EscN. In addition, the hydrophobic patch containing the chaperone–effector interacting amino acid L376 7 is mostly oriented to the inner pore but a prominent cleft observed between two subunits could probably allow the reported recognition site (Figure 9a). Moreover, the P-loop, the amino acids participating in homo-oligomerization of ATPase subunits (R198, R191), 22,29 and the amino acid for stabilization of ATP in orthologues (R349) 16 were located at the interface of the homo-hexameric model (Figure 9b). Our results show that InvCΔ79 contains two loops that undergo conformational changes upon interaction with ADP or ATPγS. The movement of these loops could be involved in the recognition of effector and chaperone proteins or in the interaction with other T3SS components including the stalk protein InvI during the dynamic process of type III secretion. 3|DISCUSSION The cytosolic interface of the membrane-embedded T3SS seems to form a hetero-complex with six or twelve ATPase subunits. 11–14,32 However, in the soluble state, the isolated full-length T3SS ATPases can form trimers, hexamers, and dodecamers. 18,19,32 It was proposed that the N-terminal FIGURE 8 Overall structural changes of InvCΔ79 in the presence of ATPγS. (a) 3D structural alignment of the apoform (blue) with the ATPγS bound form (lime). Differences in loop conformations are highlighted in rectangles and key amino acids colored as in Figure 2a. The β9-α7 loop moves a distance of approximately 8.9 Å. (b) 90rotation view showing ATPγS interaction in the ligandbinding site 1896 BERNAL ET AL.