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 InvCΔ79 dimerizes in solution in the 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.

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 β subunit 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 injectisome type III secretion system (T3SS) is a multi‐protein 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

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 molecular size of InvCΔ79 in solution. Size‐exclusion chromatography coupled to multi‐angle light scattering (SEC‐MALS) 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. Figure 1 Open in figure viewer PowerPoint 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

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/β Rossmann fold25 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 α2 and β5 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 Figure 2 Open in figure viewer PowerPoint 22 28 18 7 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).Y385 is essential for full type III secretionand the conserved E384 interacts with the stalk protein in EscN (raspberry).L376 interacts with chaperone–effector complexes (green) 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 P 6 5 P 6 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 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 C‐terminal 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. Figure 3 Open in figure viewer PowerPoint Structural analysis of InvCΔ79. (a) Structure colored by B‐factor values. Color‐coding bar shows lower (blue) to higher (red) B‐factor 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 Taken together, InvCΔ79 shows high three‐dimensional structural similarity with other T3SS ATPases including Spa47 (RMSD of 1.05 å for 325 aligned Cα residues), 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 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 ADP‐ and 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 AMP‐PNP, 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. Figure 4 Open in figure viewer PowerPoint 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

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 co‐crystallized 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. 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 β‐ and 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 hydrogen bonds with the amino acids G164 and T166, and a salt bridge with K165. The adenine group is stabilized by π‐π stacking 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 5 Open in figure viewer PowerPoint 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 Figure 6 Open in figure viewer PowerPoint Salmonella InvCΔ79 apo‐form, (b) InvCΔ79 in presence of ADP (purple) and ATPγS (lime), (c) the Shigella Spa47Δ83 with ATPγS (PDB ID: E. coli EscNΔ102 with ADP (PDB ID: 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) theInvCΔ79 apo‐form, (b) InvCΔ79 in presence of ADP (purple) and ATPγS (lime), (c) theSpa47Δ83 with ATPγS (PDB ID: 5ZT1 ) and (d) theEscNΔ102 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

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).