STRUCTURE OF THE CYTOPLASMIC DOMAIN OF THE FLAGELLAR SECRETION APPARATUS COMPONENT FLHA FROM HELICOBACTER PYLORI

Using X-Ray crystallography we have determined the structure of the cytoplasmic fragment (residues 384-732) of the flagellum secretion system protein FlhA from Helicobacter pylori at 2.4 Å resolution (R=0.224; R free =0.263). FlhA proteins and their type III secretion homologues contain an N-terminal integral membrane domain (residues 1-350), a linker segment, and a globular C-terminal cytoplasmic region. The tertiary structure of the cytoplasmic fragment contains a thioredoxin-like domain, an RNA recognition motif-like domain inserted within the thioredoxin fold, a helical domain, and a C-terminal β / α domain. Inter-domain contacts are extensive and the H. pylori FlhA structure appears to be in a closed conformation where the C-terminal domain closes against the RRM-fold domain.


INTRODUCTION
Helicobacter pylori is a motile Gramnegative ε -proteobacterium that colonises the human gastric mucosa. Infection with H. pylori causes inflammation typically resulting in gastritis, peptic and duodenal ulcer diseases and in the most severe cases, gastric adenocarcinoma (reviewed in 1,2). H. pylori is motile by means of multiple flagella that are similar in many respects to the well-studied flagella of enteric bacteria, with the exception that they are sheathed by an extension of the outer membrane and located at the cell poles (3,4). Motility is also an essential factor for colonisation and persistence of H. pylori in the gastric mucosa of the host organism (5,6).
Bacterial flagella consist of four major substructures: a basal body containing the flagellar motor and the export apparatus, a hollow rod that spans the cell envelope, a hook that functions as the torque-generating universal joint, and the flagellar filament which protrudes into the aqueous medium and provides motility (reviewed in 7,8). The basal body is composed of two connected, but distinct structures, the C-or cytoplasmic ring containing FliG, FliM and FliN which interact with both the flagellar motor (MotA and MotB proteins) and the MS (motor-stator)-ring. The MS-ring houses the export apparatus, and is composed of 24-26 copies of the ~570 residue FliF protein (9)(10)(11). The MS-ring is anchored to the cell membrane via two membrane spanning segments of FliF situated at approximately residues 23-42 and 449-460 (12). Associated with the MS-ring are the membrane-spanning proteins FlhA, FlhB, FliO, FliP, FliQ and FliR, that are thought to assemble together in the cytoplasmic membrane to make an export pore (8). The pore proteins interact with FliI and FliH, two soluble cytoplasmic components of the export apparatus, and together direct substrates to and facilitate their passage through the export pore in a defined order to ensure correct flagellum assembly. Efficient export of flagellum components requires both ATP hydrolysis, by the flagellum specific ATPase FliI, and the transmembrane protonmotive force (13,14).
Of the membrane-inserted components of the flagellar export apparatus, FlhA and FlhB are the most well characterized subunits (13,(15)(16)(17)(18)(19). FlhA encodes a ~700 residue protein that is essential for flagellum biogenesis and which contains an N-terminal integral membrane domain (amino acids 1-350) predicted to contain either six or eight membrane-spanning helices (16). A globular C-terminal domain, FlhA C (residues 350-730 of Hp FlhA), resides in the cytoplasm and likely interacts with the soluble components of the export apparatus and/or export substrates as Salmonella strains harbouring a plasmid carrying the FlhA C fragment in a wild-type background exhibit a dominant negative phenotype, including impaired motility and impaired export of rod/hook substrates (16). A number of Salmonella FlhA C point mutants have been mapped that impair motility at restrictive temperatures (19). Genetic suppressor and biochemical studies indicate that the membrane-spanning segment of FlhA interacts directly with the MS-ring protein FliF (16,18). The globular C-terminal cytoplasmic region of FliF is also known to interact with the C-Ring protein FliG (12, 20) and has been reported to interact with the cytoplasmic C-terminal fragment of FlhA in Chlamydia pneumoniae (21). Other studies show that FlhA plays an important role in keeping the flagellar export gate closed in the absence of the export regulator FliH or the flagellar ATPase FliI (13,17). The Salmonella FlhA C fragment has been well characterized (16) and crystallized (22), and a structure for Salmonella FlhA C was published online at the time of submission of this manuscript (23). The structure of the C-terminal domain of Salmonella InvA, the type III secretion homolog of FlhA, was also published online during the review of this manuscript (24).
In H. pylori, FlhA is a 733 amino acid polypeptide essential for motility (25). Deletion of FlhA or FlgM (the anti-sigma factor for σ 28 ) causes repression of the transcription of a broad range of flagellum genes (26). FlgM/FlhA double mutants have higher than normal flagellar gene expression levels for the genes down-regulated in the flhA mutant suggesting an interaction between FlhA and FlgM (26)(27)(28)(29). An interaction between H. pylori FlhA C and FlgM was recently demonstrated by a bacterial two-hybrid assay and also affinity pulldowns where purified FlgM is added to Histagged FlhA C in E. coli extracts, the interaction was however not confirmed using the two purified proteins, suggesting an additional interaction factor in E. coli extracts (29). In Salmonella, FlgM is a filament class export substrate, and is exported after hook completion. Unlike its Salmonella orthologue, H. pylori FlgM is not secreted and remains in the cytoplasm (29). Hence there are likely to be important differences in the function of FlhA in H. pylori, as compared to its role in Salmonella. Studies on FlhA from Campylobacter and other bacteria suggest functions in addition to flagellar protein export, where secretion of certain non-flagellar outer membrane proteins requires FlhA (30)(31)(32). Studies on LcrD, the Yersinia pestis type III secretion system homolog of FlhA, also demonstrate a role in the regulation of other secretion system components, suggesting this may be a general feature of FlhA homolog function (33)(34)(35). Given the high level of sequence conservation between FlhA and its type III secretion orthologs, the important central role of FlhA in regulation of H. pylori flagellum assembly and the secretion of flagellum components, and the diverse role of FlhA in the secretion of non-flagellar outer membrane proteins, we chose to study the threedimensional structure of the soluble cytoplasmic domain of FlhA (FlhA C ) from Helicobacter pylori CCUG17874.
Pfu polymerase according to the manufacturer's instructions (New England Biolabs). The fragment corresponding to amino acids 373-732 (the Cterminal Phe residue was deleted) of H. pylori FlhA was cloned into Glutathione-S Transferase gene fusion vector pGEX-6P-3 (GE Healthcare, Picatsaway, NJ) using EcoRI and BamHI restriction sites. Forward and reverse primer sequences are 5' GAC CTG GGA TCC ACA AGG GCT AAA ACC CAA GAA GAG 3', and 5' GAC GAC GAA TTC TTA GTT AAT ATG GAT CGT GCC TAA GGC. Sequencing of the insert indicated that the predicted amino acid sequence of the HP1041 gene fragment from strain 17874 differed at five positions from the published sequence from HP strain 26695. The mutations are conservative and likely have minimal impact on the tertiary structure or function of the protein.
They are: L381I, K482R, E502D, T503A, V670G, and I710V. Protein over-expression and purification-E. coli BL21 cells harbouring the pFlhA C plasmid were grown at 37°C with shaking (225 rpm) in Luria-Bertani broth, containing 100 µg/ml ampicillin. Cells were grown at 37°C to an OD 600 of 0.7 and expression of the truncated recombinant FlhA C protein was induced with 0.1 mM IPTG. After induction, cells were grown at 25°C for an additional 16-18 h. E. coli cells were harvested by centrifugation and lysed by passage twice through a French Press. The resultant supernatant was clarified by centrifugation 2X at 16,000 x g for 30 min. The Selenomethionine-labelled protein was prepared in a similar fashion, but once cells were grown to an OD 600 of 0.7 in LB, the E. coli cells were transferred to M9 minimial medium supplanted with Selenomethionine (60 mg) and the following amino acids to inhibit the E. coli methionine biosynthetic pathway (Lys, Phe, Thr (100mg); Val, Leu, Ile (50mg)). After the cells recovered for 0.5h at 37°C, they were then induced with 0.1mM IPTG and grown at 25°C for an additional 16-18 h. For both wild-type and Se-Met protein, the soluble fraction containing the recombinant GST-fusion protein was loaded onto an ÄKTA FPLC instrument, fitted with a column packed with 10 mL of glutathione Sepharose 4B resin and equilibrated in lysis buffer. After extensive washing, the GST-fusion protein was eluted using a gradient wash of reduced glutathione. The eluted GST-fusion protein fractions were pooled, dialysed overnight into cleavage buffer, and then incubated with PreScission Protease for 16 h at 4°C (GE Healthcare). The GST tag and PreScission Protease were removed by a second passage of the sample over the Glutathione sepharose column. The Cterminal domain of FlhA (FlhA C ) was further purified by ion anion exchange chromatography (Source Q) on an ÄKTA FPLC (50 mM Tris-HCl buffer pH 8.0, elution gradient of 0.0-0.5 M NaCl). FlhA C was further purified by gel filtration chromatography in 50 mM Tris buffer with 150 mM NaCl at pH 7.2 using a Superdex 200 HR 10/30 column. The purity of FlhA C was assessed after each purification step by SDS-PAGE. Protein samples were concentrated using 10 kDa molecular weight Amicon Ultra-15 Centrifugal Filter Units. Protein concentrations were measured using the Bradford dye binding assay, and/or absorbance of aromatic residues at 280 nm (ε 280 = 14,400 M -1 cm -1 ; Abs of 0.373 for 1mg/ml soln). Protein Crystallisation-Highly purified FlhA C protein was concentrated to 10 mg/mL in 20 mM Bis-Tris Propane buffer, pH 6.5, and 150 mM NaCl. The FlhA C protein was crystallised at 20° C in hanging-drop vapour-diffusion experiments. Drops contained 1.0 µl of purified protein and an equivalent volume of reservoir solution and were sealed over a reservoir filled with 1 ml of crystallisation solution. Small clusters of plate-like crystals were obtained from commercial sparse matrix screens containing ~20% (w/v) PEG-3350, 0.2 M Ca 2+ or Mg 2+ salts. Extensive optimization of the crystallization conditions yielded large, but thin plate crystals (0.5 x 0.3 x 0.03 mm) from 13.5-15% w/v mono methyl ether (MME)-PEG 5000, 100 mM Na Cacodylate buffered at pH 6.2-6.7, 0.2 M (NH 4 ) 2 SO 4 , and 6-8% v/v isopropanol. Structure determination-Crystals of Selenomet-incorporated FlhA C protein were grown as described above and were incubated in mother liquor plus 18% w/v glycerol, mounted in 20 µm nylon loops (Hampton Research), and flash frozen in a stream of liquid nitrogen gas. A Selenium Kedge MAD experiment was performed on the CMCF-I 08ID-1 beamline at the Canadian Light Source Synchrotron (Saskatoon, Canada). Diffraction data at peak, inflection and remote (low energy) wavelengths (Table 1) were then collected and processed with the HLK-2000 package (37). The crystals of the Se-Met labelled protein diffracted to a nominal resolution of 3.0 Å. The unit cell parameters fitted a C-centred Orthorhombic lattice (space group C222 1 ), with one molecule in the asymmetric unit ( Table 1). Analysis of the anomalous scattering signal from the selenium atoms, using Solve, revealed a strong signal to approximately 3.4 Å resolution (38)(39)(40). Eight of the nine Se atoms were located with Solve, the resultant Z-score was 31.5 with a figure of merit of 0.55 for all data between 50.0 and 3.00 Å resolution. A solvent-flattened electron density map (70% solvent in the unit cell) calculated to 3.0 Å resolution with Resolve clearly revealed the overall trace of the polypeptide chain, and the positions of eight of the nine expected Se-Met residues ( Figure S3). Subsequent model building was carried out with Coot (41). The initial model was refined against the Se-Met peak data using CNS (42). Native data to 2.4 Å resolution were then obtained from crystals prepared from unlabeled protein ( Table 2). This data was used for all further model refinement. Initially CNS (42) and then Refmac 5.2 were used in conjunction with R free (5% of the data) for refinement (43)(44)(45). A Ramachandran plot and representative electron density map for the final model can be found in the supplementary data. Crystallographic refinement statistics are provided in Table 2.

RESULTS AND DISCUSSION
FlhA C Tertiary Structure. The structure of the H. pylori FlhA C-terminal domain (residues 384-732) has been fully refined against diffraction amplitudes from the native crystals to 2.4 Å resolution (Figures S1 to S3) (R=22.4%, R free =26.3%). The structure contains one molecule in the crystallographic asymmetric unit and is well ordered, with the exception that helices α 6 and α 7 have higher than average thermal displacement factors. H. pylori FlhA C is composed of four domains arranged roughly at the four corners of a rectangle, and begins with a short extended fragment (residues 384 to 394) that connects the N-terminal integral membrane segment of FlhA (residues 1-350) to FlhA C (Figure 1). A Dali search (46) using the refined H. pylori FlhA C atomic coordinates revealed similarities to known protein domains ( Figure S4). The first domain resembles thioredoxin (residues 395 to 458 and 519-540 are similar to residues 16-81 of PDB 2P0J, Z-score = 6.5, rmsd 2.2 Å). Inserted in the thioredoxin-like fold is a small antiparallel β-sheet domain with the same topology of RNA recognition motifs (RRM) (residues 470 to 518) (Figures 1 and 2). The two segments connecting the thioredoxin and RRM domains each contain a proline residue (Pro469 and Pro519) that likely make this domain connection in H. pylori FlhA fairly rigid. The last helix of the thioredoxin-like domain is quite long and connects to an all-α domain (residues 541 to 614) that remotely resembles the N-terminal helical domain of ribonucleotide reductase (PDB 2R1R, Z-score = 5.5, rmsd 2.6 Å). The FlhA helical domain is in intimate contact with the FlhA C-terminal domain (residues 630 to 729). The Cterminal domain superficially resembles the βstrand arrangement and placement of two helices in the redox protein rhodanese (residues 6-95 of PDB 3G5J, Z-score = 4.6, rmsd 3.3 Å) (Figures 1  and 3). However, there is no sequence conservation between FlhA and any of these structurally similar domains.
At the juncture of the four domains of the H. pylori FlhA C molecule, there is a noticeable surface depression (Figure 1). The bottom of this depression or cleft is occupied by the β 6 -β 7 hairpin from the RRM-like domain and it contacts the α 12β 10 loop on the C-terminal domain, making van der Waals contacts between residues Leu679, Gly682, Ala684, and Pro685 of the α 12 -β 10 linker, and residues Phe494 and Met496 of the β 6 -β 7 hairpin (Figures 2 and 3). In addition, a cis-peptide bond connects residues Ala684 and Pro685 and contributes to this interaction surface ( Figure 3).
Interactions between the helical and Cterminal domains of H. pylori FlhA are mostly polar in nature, and are centred on four highly ordered salt-bridge interactions ( Figure 4). Residues making two of these salt bridges are highly conserved in FlhA sequences (Figure 2), they include Glu580 (at the end of helix α 8 ) that forms a salt bridge with Arg695 (helix α 13 ), and Glu716 (β 11 -β 12 loop) that forms a salt bridge with Arg613 (helix α 10a ). Both Glu580 and Glu716 are known sites of temperature sensitive point mutations in Salmonella FlhA (Glu547 and Glu676) that impair flagellum export function (19).
In the H. pylori FlhA C structure, the guanidinium groups of Arg613 and Arg695 are highly ordered and in close contact, effectively making a π-π stacking interaction. Other less well conserved interactions between the helical and C-terminal domains include salt bridges between Glu610 and Arg695, His579 and Glu703 (Figure 4).
Sequence and structural conservation in the FlhA/LcrD family. FlhA sequences and those of their type III secretion counterparts (InvA/LcrD etc.) are well conserved in bacteria, but are not related to any other protein families or domains. The FlhA cytoplasmic fragment sequences are less well conserved than the N-terminal transmembrane segment, and the membrane-cytoplasmic linker segment is the most poorly conserved region of the molecule (Figure 2). For FlhA C , the highest sequence conservation is found in the thioredoxin and helical domains which contain a number of invariant amino acid residues (Figure 2). In addition, conserved surface residues in these two domains tend to cluster on a fairly confined strip on one face of the FlhA C molecular surface ( Figure  1). A string of positively charged residues is part of this conserved surface patch (Figure 1).
In contrast, sequence conservation in the RRM-like domain of FlhA homologues is less extensive, especially for the type III secretion system members ( Figure 2). However, residue Trp500 in this domain is highly conserved and contacts residues in the thioredoxin-like domain. Importantly, the recently reported structure of Salmonella InvA (24) shows that the InvA RRMlike domain adopts a noticeably different conformation from the FlhA RRM-like domains in H. pylori and Salmonella FlhA structures, and contains an additional helix between strands β 5 and β 6 ( Figure 3).
The C-terminal β/α domain of FlhA is moderately well-conserved both in sequence and structure and four residues making inter-domain salt bridges with the adjacent helical domain in H. pylori FlhA C are highly conserved (Figures 2 and  4). In addition, a moderately hydrophobic surface patch on the C-terminal β/α domain of H. pylori FlhA C (secondary structure elements β 9 and α 11 plus β 10 and β 12 ), is conserved in hydrophobic character in other FlhA and type III secretion homologue sequences (Figure 2). This surface also includes a highly conserved glutamate residue (Glu641) (Figures 2 and 4). The presence of this hydrophobic surface patch on the FlhA α/β Cdomain is suggestive of a ligand binding site.
The recently published structures of Salmonella FlhA C (23) and Salmonella InvA (24) permit comparison with the structure of H. pylori FlhA C (Figures 3-4). In all three molecules, the thioredoxin-like domain, the helical domain and the C-terminal domain are very similar in tertiary structure and only differ significantly in conformation at a few insertions or deletions at surface loops (Figures 2-4). Although the RRMlike domain structure and orientation are very similar in Salmonella and H. pylori FlhA, the RRM-like domain differs significantly in structure and orientation in Salmonella InvA (24) (Figure 3). The orientation of the RRM-like domain of InvA is twisted away from the C-domain relative to FlhA when the thioredoxin-like domains are superimposed, and the InvA RRM-like domain also contains an extra helix ( Figure 3).
The highly curved N-terminal linker segment connecting the FlhA transmembrane domain to the cytoplasmic domain is also very similar in structure in H. pylori and Salmonella FlhA. This similarity in conformation of the linker segment was unexpected, as sequence conservation is minimal in the linker region and modelled thermal displacement factors are high (Figures 2-3). Hence, this suggested to us that the conformation of the linker segment and may help to precisely position the cytoplasmic domain relative to the FlhA membrane-spanning segment. If the linker conformations observed in the two FlhA crystal structures are truly reflective of the in vivo conformation of FlhA, then we can tentatively assume that the plane of the cytoplasmic membrane would be situated at the top of Figure 3. However, the linker fragment is quite short in the crystallized InvA cytoplasmic fragment and noticeably different in conformation (24).  (Figure 3). For instance, when the Nterminal two domains of H. pylori and Salmonella FlhA C molecules are superimposed by least squares, the C-terminal end of helix α 12 in Salmonella FlhA is displaced by at least 15Å relative to H. pylori FlhA. In contrast, the conformations of both the helical and C-terminal domains of Salmonella InvA and H. pylori FlhA are quite similar, and each molecule can be described as being in a closed conformation relative to Salmonella FlhA. However, the InvA molecule is closed to a greater extent than H. pylori FlhA, as the helical and C-terminal domains have undergone rigid body movements that places the α 12 -β 10 linker of the C-terminal domain closer to the RRM-like domain, facilitating interdomain interactions ( Figure 3).
Because H. pylori FlhA and Salmonella InvA adopt a similar arrangement of their four domains, the RRM-like and the C-terminal domains are in close contact in each of the two structures (24), resembling a closed conformation of the molecule (Figure 3). Although the details of this interaction differ significantly in H. pylori FlhA and Salmonella InvA (not shown), and are partly compounded by differences in the structure and conformation of the RRM-like domain, the overall mechanism of contact is similar as the α 12β 10 linker of the C-terminal β/α domain fits into a cleft between strands β 5 and β 7 of the RRM-like domain (Figure 3).
A comparison of the domain movements contributing to the open-closed structural transition suggested by the structures of the three FlhA C homologues reveals that the helical domain shifts relative to the thioredoxin domain by small structural perturbations in the vicinity of helix α 5 that links the two halves of FlhA C , resulting in a hinge-like rigid movement of the helical domain ( Figure 3). The C-domain largely moves in conjunction with the helical domain. However, there appears to be a second hinge between helices α 10a and α 10b at the juncture of the helical and C-terminal domains. The movement of α 10b relative to α 10a is further amplified by a structural rearrangement of the loop connecting helix α 10b to strand β 9 at the start of the C-terminal domain in Salmonella FlhA (Figures 3-4). The α 10a -α 10b hinge movement is most evident in Salmonella FlhA when compared to H. pylori FlhA (Figure 4). The orientations of highly conserved salt bridge residues at the interface between the FlhA C helical and C-terminal domains are also different in Salmonella FlhA, as Arg579 in Salmonella FlhA appears to be partially disordered and does not make stacking interactions with Arg653 or form hydrogen bonds with Glu676 ( Figure 4). In H. pylori FlhA (and InvA), the analogous residue, Arg613, makes stacking interactions with Arg695 and also makes two hydrogen bonds to Glu716 ( Figure 4). Hence, the conformations of these highly conserved salt bridge residues bridging the helical and C-terminal domains are essentially identical in the two closed FlhA homologue structures, but different in Salmonella FlhA which exhibits an open conformation, strongly suggesting that the precise orientations of the conserved salt bridge residues may contribute to the relative positioning of the helical and C-terminal domains and hence possibly to the maintenance of the open/closed conformation of the FlhA molecule.
Insights into FlhA/LcrD mutational data from the H. pylori FlhA structure. A number of genetic and functional studies have been published probing the function of FlhA, and in particular the FlhA C fragment from Salmonella enterica (13,(16)(17)(18)(19)21). A V404M Salmonella FlhA point mutation (Q439 in Hp FlhA) confers basal levels of motility in a normally non-motile fliH fliI null background (13,17) and lies on a highly conserved surface patch of the thioredoxin-like domain of FlhA C (Figures 2 and 3). The FlhA V404M mutation is thought to increase the likelihood of the export pore being in an open conformation, based on its ability to restore moderate levels of motility in a ΔfliHΔfliI non-motile background (13). As strains carrying flhA (V404M) are non-motile in a ΔfliI background (FliH inhibits flagellum protein export in the absence of FliI) (13,17), it is unlikely that this region of FlhA interacts with FliH. This surface of FlhA more likely interacts with an Nterminal cytoplasmic extension of the FlhB transmembrane domain as several FlhB N-terminal mutants have a very similar phenotype to the FlhA V404M mutation and residues 1-33 of FlhB most likely extend into the cytoplasm (17). The proximity of Val404 to the transmembrane linker segment in the three-dimensional structures of Salmonella and H. pylori FlhA C is consistent with this interpretation. Gln439 (equivalent to Salmonella Val404) is also near a modest dimer interface in the H. pylori FlhA C crystal structure (Supplemental Figure 5). The significance of the dimer interface in the H. pylori FlhA C crystal is not clear, as the protein elutes as a monomer on analytical gel filtration columns.
Several temperature-sensitive Salmonella FlhA C missense mutants that cannot regrow flagella at the restrictive temperature have been reported (19). These mutants have been extensively analyzed with the recently reported Salmonella FlhA C structure and will not be discussed further (23,47). However the positions of the mutations are indicated in Figure 2, and two of the mutations (S. t. E547K and E676K) have already been mentioned in relation to the conserved salt bridges at the interface between the helical and C-terminal domains (Figures 2 and 4).
The phenotypes of eighteen 20-residue deletions (Δ1:328-347 through to Δ18: 668-687) spanning Salmonella FlhA C have also been described (16). All but one of these deletion mutants partially complemented the loss of motility when introduced into a flhA null background. However, the Δ18 deletion, corresponding to removal of the last two β-strands of FlhA C (Figure 2 and 5) did not restore motility in a flhA null strain (16). The relative severity of the Δ18 deletion suggests the deleted region is critical for FlhA function. Two other Salmonella FlhA deletion mutants identified in the same study (Δ6, 428-447 and Δ17, 648-667) were reported to be dominant negative for motility when introduced into fla + strains ( Figure 2) (16). Interpretation of the dominant FlhA C deletions, however is difficult within the context of the many other non-dominant FlhA deletions that would also likely severely disrupt the FlhA C structure.
Mutational screens of the Yersinia pestis type III secretion system have revealed the importance of the FlhA orthologue LcrD in controlling the transcription of other Type III export components, a feature that these proteins have in common with FlhA from H. pylori (27,28,(33)(34)(35). An LcrD nonsense mutation (Q574Stop) truncates FlhA C at the end of helix α 9 , hence deleting helices α 10a and α 10b and the entire C-terminal domain (Figures 2 and 5) (35). The resultant truncated LcrD protein was defective in export substrate transcription and secretion, presumably through the cytoplasmic retention of the unidentified co-repressor of LcrH (35). An LcrD point mutant with a similar secretion defective phenotype (LcrD Y670C) (35) maps to position 697 on helix α 13 of Hp FlhA ( Figure 5).
In the flagellum system, null mutations in the Salmonella flk gene permit premature switching from rod/hook substrates to filament type substrates, either in a fla + background, or in a non-motile strain that lacks an outer rod and secretes flagellum components directly into the periplasm (48). Two nonsense mutations in FlhA at Q588 or Q589 (N622 or A623 in Hp FlhA) that cleanly delete the FlhA C C-terminal domain yielded a phenotype similar to the flk null in a temperature sensitive screen. A third early switching mutant corresponded to an internal deletion of helices α 11 through to the end of α 12 (ΔI600-A644; ΔL634-I683 in Hp FlhA) (Figures 2  and 5). The truncated Salmonella FlhA mutants permit early secretion of FlgM (a filament type substrate and anti σ 28 factor) at permissive temperatures, however, secretion of FlgM into the periplasm is not observed at the restrictive temperature (48). These FlhA truncation mutants also disrupt flagellar export when introduced to a fla + background (48). A surprising feature of the early switching FlhA truncation mutations is that they overlap almost exactly with the Yersinia pestis LcrD truncation that is defective in type III effector secretion (Figures 2 and 5) (34).
The phenotype of the flhA early secretion mutations suggests that the C-terminal domain of Salmonella FlhA not only prevents premature secretion of filament class substrates but also facilitates the secretion of rod/hook substrates, as the same mutants typically do not complete rod/hook structures in an otherwise wild type background. Although the flk gene is likely not present in H. pylori or other epsilon proteobacteria, it is tempting to speculate that the C-terminal domain of H. pylori FlhA will function similarly to Salmonella FlhA in substrate switching and protein export due to the high level of sequence conservation for FlhA, FlhB and FliK. Furthermore, the finding of similar mutants in Yersinia pestis LcrD with secretion defective phenotypes suggest a common mechanism of FlhA homologue function in both flagellar and type III secretions systems. Nevertheless, there are likely important differences for FlhA function in H. pylori, as FlgM remains cytosolic during normal flagellum function, FlhA and FlgM appear to interact with each other, likely via an unknown third partner protein (29), and FlhA or FlgM null mutants have dramatic global effects on flagellum gene expression. (27,28).    Structurally-based alignment of FlhA C and type III secretion homologue sequences. Calculated with T-Coffee (51) and rendered using Espript (52). Secondary structure assignments for the X-Ray structure of H. pylori FlhA C are shown, and labeled according to the Salmonella FlhA C structure (23). Highly conserved residues are boxed in red, moderately conserved residues in pink. Salmonella temperature sensitive secretion point mutations in FlhA C are marked with purple stars (19). Salmonella flhA nonsense mutations (Q588Stop, Q589Stop) that permit early secretion of FlgM are marked with red stars (48). The Salmonella FlhA V404M mutation is marked with an orange square (13,17). Yersinia pestis LcrD secretion defective mutants are marked with triangles, red indicates a nonsense mutation at Q574 that deletes the C-terminal domain, pink depicts a Y670C missense mutation (34,35). Conserved salt bridge interactions between the helical and C-domains of H. pylori FlhA C are marked with red (Glu) or blue (Arg) squares. Green circles mark residues that make up a predominantly hydrophobic surface on the C-domain. Salmonella FlhA deletion mutants mentioned in the text are marked by colored lines (16,48).    (34,35,48). The position of an internal Salmonella FlhA deletion (Δ 600-644) that permits early secretion of filament class substrates is also shown in blue (48). Table 1 Data collection, phasing, and structure refinement statistics for H. pylori FlhA C .

Data collection statistics a,b
Se-Met Native (C222 1 ) Peak Inflection Remote Unit cell parameters (a, b, c) (Å) Residues in allowed regions (%) e 8.3 a R work = Σ hkl | |F obs (hkl)| |−|F calc (hkl)| | / Σ hkl |F obs( hkl)|, where |F obs (hkl)| and |F calc (hkl)| are the observed and calculated amplitudes, respectively, for the structure factor F(hkl). b R free is the equivalent of R work for 5% of the reflections (randomly selected) which were not used in structure refinement. c Values in parentheses correspond to the highest resolution shell. d B-factor and r.m.s.d values were calculated with Refmac as implemented in CCP4 (43)(44)(45). e The Ramachandran plot was generated with Procheck in CCP4 (See Supplemental Figure S3) (45,53).
by guest on March 24, 2020