Author response: Bacterial flagellar capping proteins adopt diverse oligomeric states (2016)

Article Figures and data Abstract eLife digest Introduction Results Discussion Materials and methods Data availability References Decision letter Author response Article and author information Metrics Abstract Flagella are crucial for bacterial motility and pathogenesis. The flagellar capping protein (FliD) regulates filament assembly by chaperoning and sorting flagellin (FliC) proteins after they traverse the hollow filament and exit the growing flagellum tip. In the absence of FliD, flagella are not formed, resulting in impaired motility and infectivity. Here, we report the 2.2 Å resolution X-ray crystal structure of FliD from Pseudomonas aeruginosa, the first high-resolution structure of any FliD protein from any bacterium. Using this evidence in combination with a multitude of biophysical and functional analyses, we find that Pseudomonas FliD exhibits unexpected structural similarity to other flagellar proteins at the domain level, adopts a unique hexameric oligomeric state, and depends on flexible determinants for oligomerization. Considering that the flagellin filaments on which FliD oligomers are affixed vary in protofilament number between bacteria, our results suggest that FliD oligomer stoichiometries vary across bacteria to complement their filament assemblies. https://doi.org/10.7554/eLife.18857.001 eLife digest Many bacteria, including several that cause diseases in people, have long whip-like appendages called flagella that extend well beyond their cell walls. Flagella can rotate and propel the bacteria through liquids, such as water or blood, and they are constructed primarily from thousands of copies of a single protein called flagellin. When flagella are built, the flagellin proteins are placed in their proper positions by another protein called FliD, several copies of which form a cap on the end of flagella. Without FliD, bacteria cannot properly assemble flagella and, thus, can no longer swim; this also hinders their ability to cause disease. Determining the three-dimensional structure of a protein, down to the level of its individual atoms, can provide unique insights into how the protein operates. However, no one had resolved the structure of a FliD protein from any bacterium to this level of detail before. Now, Postel et al. report the high-resolution structure of a large fragment of FliD from the bacterium Pseudomonas aeruginosa. The structure reveals that parts of this FliD protein are shaped like parts of other proteins from which flagella are constructed, including the flagellin protein that FliD places into position. Some parts of the FliD protein are also very flexible and these parts of the protein are responsible for holding numerous FliD proteins together as a cap. Finally, Postel et al. saw that six copies of FliD bind to one another to form a protein complex on the end of flagella. This last finding was particularly unexpected since it was thought that all FliD proteins formed five-membered cap complexes, an assumption that was based largely on studies of FliD from another bacterium called Salmonella. The current structure covers about half of the FliD protein, and so the next challenge is to determine the structure of the full-length protein. An improved understanding of the structure of FliD may, in future, help researchers to design drugs that stop bacteria from building flagella and, therefore, from swimming and causing disease. https://doi.org/10.7554/eLife.18857.002 Introduction Pathogenic bacteria cause a multitude of deadly human diseases. Many of these microbes possess flagella, molecular machines responsible for cell motility, adherence to host cells, and pathogenicity (Duan et al., 2013; Haiko and Westerlund-Wikstrom, 2013). Flagella are helix-shaped hollow attachments formed predominantly by thousands of copies of the protein flagellin (also called FliC), anchored in the bacterial membrane by a hook (or joint) that is attached to the basal body and that is composed of rotary motor proteins (Arora et al., 1998). A proton motive force typically drives the propeller motion of flagella (Berg, 2003), resulting in swimming motility. A FliD (also called HAP2) oligomer forms the cap protein complex that is located at the tip of the flagellar filament (Yonekura et al., 2000). This complex controls the distal growth of the filament by regulating the assembly of FliC molecules, which are transported through the hollow filament from the cytoplasm to the tip of the flagellum. The dynamic movement of FliD in this assembly was modeled based on low (~26 Å)-resolution cryo-electron microscopic (EM) structures of the Salmonella serovar Typhimurium flagellum-cap complex (Maki-Yonekura et al., 2003; Yonekura et al., 2000, 2003), which adopts the shape of a five-legged stool with flexible leg domains that regulate the assembly of new FliC molecules onto the tip of the growing flagellum (Maki-Yonekura et al., 2003). It has been suggested that the plate of the stool is formed by core regions of the FliD molecule, and that disordered/flexible regions form the five leg structures (Vonderviszt et al., 1998) that are known to interact with the FliC filament. FliD exhibits low sequence similarity to the flagellar hook proteins and to FliC. Nevertheless, it shares the disordered terminal regions of these flagellar proteins, a common structural characteristic that is thought to control the polymerization of flagellar proteins and to play an important role in interaction with the FliC filament (Vonderviszt et al., 1998). These regions are the most conserved in FliD sequences across bacteria. Flagellum-mediated motility is crucial for the virulence and pathogenicity of numerous bacteria, including Campylobacter jejuni (Black et al., 1988), Salmonella (Allen-Vercoe and Woodward, 1999; Marchetti et al., 2004), Escherichia coli (La Ragione et al., 2000), Vibrio cholera (Krukonis and DiRita, 2003), and Pseudomonas aeruginosa (Arora et al., 2005), as well as the major causative agent of gastric cancer Helicobacter pylori (Kim et al., 1999). To date, however, no high-resolution structure of any FliD protein exists. To better define the roles of FliD in bacterial motility and pathogenesis, we determined the first X-ray crystal structure of FliD at 2.2 Å resolution, and assessed the structural contributions of its flexible regions using a multitude of complementary biophysical and functional analyses. Results Crystal structure of the FliD protein from P. aeruginosa PAO1 To facilitate crystallization of FliD from the P. aeruginosa PAO1 strain, we deleted the predicted coiled-coil domains on both the N- and C-termini of full length FliD, which has 474 residues (FliD1–474), to generate the truncated FliD78-405 (Figure 1a, Figure 1—source data 1). We expressed FliD78–405 in E. coli with an N-terminal His6-tag and purified it to homogeneity by Ni2+-NTA, size exclusion and anion exchange chromatography. We improved initially weakly diffracting crystals of FliD78–405 by random matrix microseed screening (Bergfors, 2003), yielding crystals that diffracted to 2.2 Å resolution. In the absence of any homologous protein that could be used as a model for molecular replacement, we crystallized a seleno-methionine derivative of FliD78–405 that included four leucine-to-methionine mutations (FliD78–405/L4–M4). This crystal provided phase information sufficient to build an initial model, which we used subsequently for molecular replacement with the native FliD78–405 dataset (Figure 1—source data 1). We modeled residues 80–273 into clear electron density, including all side chains, but observed density of increasingly poor quality in the C-terminus beyond residue 273 (Figure 1—figure supplement 2a). Thus, we were able to model with confidence only a single α helix in this region, corresponding to residues 274–308, with incomplete side chain structures. To determine whether the remaining region of the protein actually existed in the crystals and not just in the protein preparation used for crystallization, we analyzed crystals using liquid chromatography-mass spectrometry (LC-MS) and SDS-PAGE. Both analyses indicated that the crystals consisted of an approximate 50:50 mixture of the FliD78–405 protein used for crystallization and a further proteolyzed version with a molecular weight of about 27 kDa. The N-terminal His6-tag is still detectable by Western blot (Figure 1—figure supplement 2b). Thus, the proteolyzed form corresponds approximately to residues 78–319 of FliD. The 86 residues absent from the C-terminus in a population of FliD proteins are clearly not required for crystal packing, suggesting that they are highly flexible even in a crystalline environment. Figure 1 with 2 supplements see all Download asset Open asset Crystal structure of Pseudomonas FliD reveals structural similarity to other flagellar proteins. (a) Schematic representation of the FliD proteins used in these studies. Protein domain/region boundaries are labeled and are drawn approximately to scale. (b) Crystal structure of the Pseudomonas FliD78–405 monomer subunit with spectrum coloring from the N-terminus (blue) to the C-terminus (red). Head domain 1, head domain 2 and the leg region are indicated. (c) Superposition of the FliD78–405 crystal structure (domain coloring as in panel (a)) and Burkholderia FlgK/HAP1/hook filament capping protein (cyan). (d) Superposition of the FliD78–405 crystal structure (domain coloring as in panel (a)) and Pseudomonas flagellin/FliC (magenta). https://doi.org/10.7554/eLife.18857.003 Figure 1—source data 1 Crystallographic data collection, phasing and refinement statistics. https://doi.org/10.7554/eLife.18857.004 Download elife-18857-fig1-data1-v2.xlsx FliD is structurally similar on the domain level to FliC and FlgK Our crystal structure of FliD78–405 reveals that it consists of two discreet regions with distinct conformational properties, corresponding to a stable head region and a flexible and/or disordered leg region (Figure 1b). The head region is itself comprised of two separate, but entwined, protein domains. Residues 80–101 form two β strands that belong to the first domain (Head 1), the second domain (Head 2) is formed in its entirety by the contiguous residues 104–230, and residues 231–273 then complete the first domain. The second domain is, thus, a loop insertion of the first domain. We searched for structural homologs of these domains in the Protein Data Bank and found that the first domain of the head region (Head 1) exhibits high structural similarity (RMSD=2.5 Å), despite low sequence identity (14%), to the FlgK/HAP1/hook filament capping protein of Burkholderia pseudomallei (PDB code 4UT1; Figure 1c). Likewise, the second head region domain (Head 2) exhibits high structural similarity (RMSD=2.7 Å), despite low sequence identity (17%), to the FliC/flagellin protein of P. aeruginosa (PDB code 4NX9; Figure 1d). In contrast to the head region, the leg region of FliD is highly flexible, as indicated by the paucity of electron density corresponding to residues 274–405 (Figure 1—figure supplement 2a). Despite this, we were able to model the initial α helical structural element, corresponding to residues 274–308, of this region. This helix extends from the axis of the head region at a nearly perpendicular angle, resulting in an L-shaped monomer subunit structure (Figure 1b). FliD from P. aeruginosa PAO1 forms a hexamer In the crystal, FliD78–405 monomer subunits are arranged in hexamers, resulting in a shape akin to a six-pointed star when viewed from the top of the FliD oligomer (Figure 2a), which corresponds to the distal end of the growing flagellum. This star shape has a minimum inner diameter of 48 Å and a maximal outer diameter of 136 Å. When viewed from the side (Figure 2b), the FliD hexamer appears as a six-legged stool, the legs of which extend 55 Å below the bottom of the head region. Additional crystallographic symmetry results in the stacking of hexamers in alternating head-to-head and leg-to-leg orientations (Figure 2c). The leg-to-leg stacking forms dodecamers, resulting from the helix–helix interaction of the residues 274–302 of stacked molecules and the interaction of residues 303–308 of one FliD78–405 molecule with Head 1 domain of a stacked molecule, burying a surface area of 1362 Å2. The formation of dodecamers may be unique to the FliD78–405 fragment, as this strand could potentially be replaced by additional N-terminal residues in the full-length FliD1–474 protein. All of the morphologies observed for Pseudomonas FliD are highly reminiscent of the pentamer/decamer oligomeric organization of Salmonella FliD as determined by low-resolution cryo-EM analysis (Maki-Yonekura et al., 2003) (Figure 2d). Indeed, despite the difference in the stoichiometries of the Pseudomonas and Salmonella FliD oligomers, the gross measurements are nearly identical for these two proteins of similar molecular weight. In our crystal structure, Pseudomonas FliD measures 136 Å in diameter with a head region that is 30 Å deep and a leg region that is 55 Å long; whereas, in the cryo-EM structure, Salmonella FliD measures 145 Å in diameter with a head region that is 30 Å deep and a leg region that is 55 Å long (Maki-Yonekura et al., 2003). Figure 2 Download asset Open asset Pseudomonas FliD forms hexamers in crystals. (a) Top view, cartoon representation of the FliD78–405 hexamer. Each monomer subunit is colored distinctly and inner diameter dimension is indicated. (b) Side view, cartoon representation of the FliD78–405 hexamer. Each monomer subunit is colored distinctly. Outer dimensions are indicated. (c) FliD78–405 hexamers as arranged in the crystal are stacked head-to-head and leg-to-leg (shown) in an alternating fashion, with residues 303–308 assembling in the Head 1 domain of an opposing molecule (close-up views) leading to a dodecameric crystal packing. (d) Cryo-EM structure of Salmonella FliD from (Maki-Yonekura et al., 2003) for comparison. https://doi.org/10.7554/eLife.18857.007 The stoichiometry of Pseudomonas FliD differs from that of Salmonella FliD and our Pseudomonas FliD crystals belong to the P6 space group, which could possibly force a non-physiological oligomeric organization of subunits. Thus, we confirmed that the hexameric assembly of FliD78–405 occurs not only in the crystalline environment but also in solution using negative stain EM class averaging (Tang et al., 2007), resulting in an image that clearly exhibits a hexameric assembly (Figure 3a). These data are consistent with the oligomerization state that we detected in the crystal structure, in which a view looking down the α-helices of the leg reveals a six-membered ring organization. We also verified that both FliD78–405 and full-length FliD1–474 form oligomers by both analytical ultracentrifugation (AUC) and crosslinking analyses. We found that the FliD78–405 fragment that we crystallized oligomerizes up to a dodecameric state (Figure 3b,c), similar to the crystallographic assembly (Figure 2c). Additionally, we collected small-angle X-ray scattering (SAXS) data of FliD78–405, for which the calculated radial distribution function (Figure 3d) is characteristic of an oligomeric assembly forming a hollow sphere (Svergun and Koch, 2003). These data produce a molecular envelope that superimposes well with our dodecameric FliD78–405 X-ray crystal structure (Figure 3d). Full-length FliD1–474, by contrast, forms up to hexamers in solution (Figure 3e,f), which are likely to represent the physiologically relevant oligomerization state of this protein on the tip of the flagellum. The predominant tetrameric species in solution identified by AUC may be a stable intermediate on the path to hexamer formation (Figure 3e). Figure 3 Download asset Open asset Pseudomonas FliD oligomerization. (a) Negative stain EM image of FliD78–405: left, single particles (scale bar=1000 Å); right, class-averaged particle (scale bar=50 Å). (b) AUC analysis of FliD78–405 proteins at pH 8.0 (upper panel) and pH 5.0 (lowel panel) indicates that it forms dodecamers in solution. (c) Silver-stained SDS-PAGE after chemical crosslinking of FliD78–405. (d) SAXS analysis of FliD78–405. Kratky plot (I*q2 versus q) and radial distribution function calculated by GNOM, and SAXS envelopes calculated by DAMMIF, with superimposed crystal structures are shown for FliD78–405 at 10.7 mg/mL (red, used to calculate envelope), 5.4 mg/mL (blue) and 2.7 mg/mL (grey). (e) AUC analysis of full length FliD1–474 proteins at pH 8.0 (upper panel) and pH 5.0 (lower panel) indicates a mixture of oligomers, including tetramers and hexamers. (f) Silver-stained SDS-PAGE after chemical crosslinking of FliD1–474. https://doi.org/10.7554/eLife.18857.008 As the hexameric assembly of full-length Pseudomonas FliD1–474 proved to be unstable in solution in the absence of the flagellar filament, we sought to stabilize it using our newfound understanding of its structure. We used our crystal structure of FliD78–405, as input to Disulfide by Design 2.0 (Craig and Dombkowski, 2013), to identify cysteine mutations that would lead to a stable, disulfide-bridged hexameric FliD1–474. We found that when two residues within neighboring head domain subunits, I167 and D253, were each mutated to a cysteine residue (Figure 4a) a stable, hexameric full-length FliD1–474(I167C/D253C) resulted under non-reducing conditions, as shown by SDS-PAGE (Figure 4b) and SAXS (Figure 4c) analyses. We also confirmed that the cysteine bridges resulted in the expected interfaces between head domains by employing mass spectrometry to compare the peptide coverage under reducing and non-reducing conditions (Figure 4—figure supplement 1a) and by successfully detecting the correct cysteine bridges (C167–C253) while ruling out non-specific cysteine bridging (C167–C167 and C253–C253) (Figure 4—figure supplement 1b–e). Figure 4 with 3 supplements see all Download asset Open asset Stable hexameric DM1-FliD1–474 complements P. aeruginosa PAO1 dFliD transposon strain. (a) Location of residues I167 and D253, which were predicted by the web server Disulfide by Design 2.0 (Craig and Dombkowski, 2013) to form stable disulfide bridges after mutation to cysteines. (b) FliD1–474(I167C/D253C) analyzed under reducing (lane 1) and non-reducing (lane 2) conditions by SDS-PAGE. (c) SAXS analysis of FliD1–474(I167C/D253C). Kratky plot (I*q2 versus q) and radial distribution function calculated by GNOM for 9.75 mg/mL (blue, used to calculate envelope), 4.88 mg/mL (red) and 2.44 mg/mL (grey). SAXS envelope calculated by DAMMIF with superimposed FliD78–405 crystal structure. (d) Swimming motility assay of wildtype PAO1 (WT), FliD transposon strain PW2975 (ΔfliD), ΔfliD complemented with FliD1–474 (ΔfliD/fliD1–474) or FliD1–474(I167C/D253C) (ΔfliD/fliD1–474(I167C/D253C)), respectively. (e) Western blot using anti-FliD scFv-Fc SH1579-B7 showing purified protein FliD1–474(I167C/D253C) under reducing (lane 1) and under non-reducing (lane 2) conditions. The presence of FliD in flagella preparations from wildtype PAO1 (lane 4), ΔfliD (lane 5), ΔfliD/fliD1–474(lane 6) and ΔfliD/fliD1–474(I167C/D253C) (lane 7) was analyzed under non-reducing conditions. The molecular weight standard is shown in lane 3 and the corresponding molecular weights are indicated on the right side of the blot. The 50 kDa and the 300 kDa bands representing FliD1–474 or hexameric FliD1–474(I167C/D253C), respectively, are indicated by red arrows. https://doi.org/10.7554/eLife.18857.009 To show that the hexameric form of Pseudomonas FliD is functional in vivo, we complemented the fliD transposon strain PW2975 (ΔfliD) with wildtype fliD1–474 and hexamer-stabilized fliD1–474(I167C/D253C), resulting in Pseudomonas PAO1 strains ΔfliD/fliD1–474 and ΔfliD/fliD1–474(I167C/D253C), respectively. We found that swimming motility that was lost in the ΔfliD strain was restored in both ΔfliD/fliD1–474 and ΔfliD/fliD1–474(I167C/D253C) complementation strains, similar to our observations in the wildtype Pseudomonas PAO1 strain (Figure 4d). Using antibodies that we generated by phage display to Pseudomonas PAO1 FliD, we confirmed the expression of full-length FliD proteins by Western blot analysis from preparations of flagella isolated from live bacteria from both ΔfliD/fliD1–474 and ΔfliD/fliD1–474(I167C/D253C) complementation strains (Figure 4e). In flagella preparations from the ΔfliD/fliD1–474(I167C/D253C) complementation strain, the FliD1–474(I167C/D253C) protein produced by Pseudomonas maintains its hexameric oligomeric state (Figure 4e). In contrast, subsequent to flagella isolation from live bacteria, FliD1–474 does not maintain a stable hexameric complex in either the wildtype Pseudomonas PAO1 strain or in the ΔfliD/fliD1–474 complementation strain (Figure 4e). The ΔfliD transposon strain does not form flagella, as indicated by the lack of FliC in the analyzed flagella preparations (lane 5 on the stained Western blot membrane, Figure 4—figure supplement 2). In comparison, the wildtype and all complementation strains form flagella as indicated by the presence of flagellin/FliC in the purified flagella samples (Figure 4—figure supplement 2). Together, these data that FliD that is in its hexameric assembly can form functional flagella that Pseudomonas bacteria to like Pseudomonas with wildtype FliD. Thus, the hexamer oligomeric state of Pseudomonas FliD is functional in We also whether Salmonella FliD, which is known to form when capping the flagellar filament, could function as a capping protein for Pseudomonas flagella. In contrast to the in functional hexameric forms of P. aeruginosa complementation of the PAO1 PW2975 transposon strain with fliD from Salmonella not swimming motility (Figure 4—figure supplement As this was for expression in Escherichia we also confirmed that a wildtype PAO1 full-length FliD1–474 by a swimming motility in the ΔfliD strain Figure 4—figure supplement other oligomeric state that could the of Salmonella FliD to complement these data suggest that Pseudomonas flagella may FliD proteins that hexameric of the head domains FliD oligomerization the FliD78–405 crystal structure exhibits between the head regions of FliD78–405 subunits the each of these interfaces is with a surface area of only and (Figure to and To determine whether these were sufficient to oligomerization of FliD, we expressed and purified the head region AUC (Figure chemical crosslinking (Figure and SAXS analysis (Figure we observed that when FliD the leg region and the N- and coiled-coil it is predominantly in the form of to a in but to form oligomers as the longer of FliD that we conditions, including in have been shown to the polymerization of flagellar filaments et al., and capping proteins et al., we additional AUC and found that is at pH 8.0 and approximately at pH we observed no oligomers of of conditions (Figure We also by chemical the oligomerization of FliD only the N-terminal coiled-coil domain Figure or the coiled-coil domain Figure We found to be with a of species to the oligomerization Kratky and radial distribution calculated from SAXS data of the either the N- or coiled-coil or respectively, that these proteins with flexible regions that are clearly in the resulting molecular envelopes (Figure These data that the force for of Pseudomonas FliD in molecular determinants of the head region and, at a residues in the N-terminal and coiled-coil domains. FliD78–405 is the and N-terminal coiled-coil domain but still into dodecamers, as shown in the crystal structure, and which is likely by strand replacement in the head region domain 1 and helix–helix interaction of stacked molecules (Figure 2c). Figure 5 Download asset Open asset determinants of Pseudomonas FliD oligomerization of the stable head region. (a) formed between head region monomer subunits, with an of the Head domain 1 is domain 2 is and are red and respectively. (b) AUC analysis of the head region at pH 8.0 (upper panel) and pH 5.0 (lower panel) reveals a species at pH 8.0 and the additional presence of a species at pH (c) Silver-stained SDS-PAGE after chemical crosslinking of (d) SAXS analysis of Kratky plot (I*q2 versus q) and radial distribution function calculated by GNOM and SAXS envelopes calculated by DAMMIF with superimposed crystal structures are shown for at mg/mL mg/mL (red, used to calculate the and mg/mL (e) Silver-stained SDS-PAGE after chemical crosslinking of (f) Silver-stained SDS-PAGE after chemical crosslinking of Figure with 1 supplement see all Download asset Open asset X-ray scattering (SAXS) data of and FliD1–474. SAXS Kratky versus radial distribution function calculated by GNOM and SAXS envelopes calculated by DAMMIF are shown (a) at mg/mL mg/mL (red) and mg/mL (b) at mg/mL mg/mL (red) and mg/mL and (c) FliD1–474 at mg/mL mg/mL 2.7 mg/mL mg/mL and mg/mL The N- and regions of FliD are highly flexible A large of FliD sequence to high-resolution structural including Pseudomonas FliD residues and we spectrometry with FliD78–405 to define its regions and to its dynamic We FliD78–405 to exchange for to 2 and observed that the head region of FliD78–405 adopts a largely stable with observed for domain 2 to that of domain The leg region, particularly residues to the α helix observed in the crystal structure, is disordered or stable (Figure Residues head domain 1 to the leg helix display as indicated by et al., that in envelopes (Figure The FliD78–405 protein used in these was properly as shown by as were all other FliD protein that we produced (Figure supplement 1). When to our crystal structure of FliD78–405, the of exchange on the peptide level corresponds to the of conformational on the residue level (Figure When we used a difference plot to compare the of exchange of full length FliD1–474 with that of FliD78–405, we observed that residues in FliD78–405 (Figure this of residues in head domain 2 are which form the between the two head region domains in the hexameric complex (Figure We observed an additional region of for residues of residues to the β strand replacement in the opposing head region domain 1 that drives formation of FliD78–405 (Figure Kratky calculated from SAXS data of full-length FliD1–474 at pH which is under these conditions (Figure supplement 1), the flexible of this protein. molecular envelopes calculated from SAXS data collected at of FliD1–474 also conformational (Figure Figure with 1 supplement see all Download a