Bacterial flagellar capping proteins adopt diverse oligomeric states

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.

Keywords: Pseudomonas; X-ray crystallography; analytical ultracentrifugation; biophysics; flagella; hydrogen-deuterium exchange; infectious disease; microbiology; structural biology.

Figure 1.. 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 FliD_78–405 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 FliD_78–405 crystal structure (domain coloring as in panel (a)) and Burkholderia FlgK/HAP1/hook filament capping protein (cyan). (d) Superposition of the FliD_78–405 crystal structure (domain coloring as in panel (a)) and Pseudomonas flagellin/FliC (magenta). DOI: http://dx.doi.org/10.7554/eLife.18857.003

Figure 1—figure supplement 1.. Protein sequence of FliD_1–474.

The protein sequence of FliD from P. aeruginosa PAO1 is shown. The tertiary domain structure based on the presented X-ray crystal structure and the predicted secondary structure is indicated. DOI: http://dx.doi.org/10.7554/eLife.18857.005

Figure 1—figure supplement 2.. Electron density and protein degradation of FliD crystals.

(a) The overall electron density observed in the crystal structure of FliD is shown. Close-up views of representative residues in the head 1, head 2 and leg regions reveal low-quality and missing side-chain density in the leg region in comparison to the well-structured head domains. (b) SDS-PAGE (lane 1) and anti-His₆-horseradish peroxidase (lane 2) Western blot analysis reveal C-terminal degradation of FliD_78–405 in crystals. LC-MS analysis indicates the presence of the native FliD_78–405 protein and degradation products in crystals of FliD_78–405. DOI: http://dx.doi.org/10.7554/eLife.18857.006

Figure 2.. Pseudomonas FliD forms hexamers in crystals.

(a) Top view, cartoon representation of the FliD_78–405 hexamer. Each monomer subunit is colored distinctly and inner diameter dimension is indicated. (b) Side view, cartoon representation of the FliD_78–405 hexamer. Each monomer subunit is colored distinctly. Outer dimensions are indicated. (c) FliD_78–405 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. DOI: http://dx.doi.org/10.7554/eLife.18857.007

Figure 3.. Pseudomonas FliD oligomerization.

(a) Negative stain EM image of FliD_78–405: left, single particles (scale bar=1000 Å); right, class-averaged particle (scale bar=50 Å). (b) AUC analysis of FliD_78–405 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 FliD_78–405. (d) SAXS analysis of FliD_78–405. 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 FliD_78–405 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 FliD_1–474 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 FliD_1–474. DOI: http://dx.doi.org/10.7554/eLife.18857.008

Figure 4.. Stable hexameric DM1-FliD_1–474 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) FliD_{1–474(I167C/D253C)} analyzed under reducing (lane 1) and non-reducing (lane 2) conditions by SDS-PAGE. (c) SAXS analysis of FliD_{1–474(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 FliD_78–405 crystal structure. (d) Swimming motility assay of wildtype PAO1 (WT), FliD transposon strain PW2975 (ΔfliD), ΔfliD complemented with FliD_1–474 (ΔfliD/fliD_1–474) or FliD_{1–474(I167C/D253C)} (ΔfliD/fliD_{1–474(I167C/D253C)}), respectively. (e) Western blot using anti-FliD scFv-Fc SH1579-B7 showing purified protein FliD_{1–474(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/fliD_1–474(lane 6) and ΔfliD/fliD_{1–474(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 FliD_1–474 or hexameric FliD_{1–474(I167C/D253C),} respectively, are indicated by red arrows. DOI: http://dx.doi.org/10.7554/eLife.18857.009

Figure 4—figure supplement 1.. Analysis of FliD_{1–474(I167C/D253C)} peptides following pepsin digestion under reducing and non-reducing conditions.

(a) Comparison of peptide coverage maps determined by LC/MS after pepsin digestion in the presence or absence of TCEP. Shown in light blue are the peptides that are identified only after treatment with TCEP, in dark blue are peptides identified both in the presence and in the absence of TCEP. The introduced cysteine residues are highlighted in red. (b) Peptides containing the introduced cysteines that were identified by Biopharmalynx in the presence and/or absence of TCEP are tabulated along with retention time (RT), and the number and percentage of b/y ions identified. (c–e) b/y fragment ions for three peptides surrounding the introduced cysteines as identified by Biopharmalynx in the presence and absence of TCEP. (c) and (e) show the b/y ions for peptides 165–176 and 251–257. (d) shows the b/y ions identified for the disulfide-bridged peptide 165–176 x 251–257. DOI: http://dx.doi.org/10.7554/eLife.18857.010

Figure 4—figure supplement 2.. Western blot analysis of PAO1 strain flagella preparations.

Coomassie-stained membrane of the Western blot displayed in Figure 4e showing purified protein FliD_{1–474(I167C/D253C)} under reducing (lane 1) and under non-reducing (lane 2) conditions. The presence of FliC in flagella preparations from wildtype PAO1 (lane 4), ΔfliD (lane5), ΔfliD/fliD_1–474(lane 6) and ΔfliD/fliD_{1–474(I167C/D253C)}(lane 7) was analyzed under non-reducing conditions. The molecular weight standard is shown in lane 3 and the molecular weights are listed on the right side of the membrane. The 50 kDa FliC bands are indicated by black arrows. DOI: http://dx.doi.org/10.7554/eLife.18857.011

Figure 4—figure supplement 3.. Swimming motility assay.

Swimming motility of wildtype PAO1 (WT), FliD transposon strain ΔfliD, ΔfliD complemented with FliD_1–474 with an optimized codon usage for Echerichia coli expression (ΔfliD/fliD_PAOfliDe) or Salmonella typhimurium FliD_1–467 with an optimized codon usage for E. coli expression (ΔfliD/fliD_StyFliDe). DOI: http://dx.doi.org/10.7554/eLife.18857.012

Figure 5.. Molecular determinants of Pseudomonas FliD oligomerization reside outside of the stable head region.

(a) Intermolecular interface formed between head region monomer subunits, with an 'open book' rendering of the interface expanded below. Head domain 1 is yellow; domain 2 is orange; interface oxygen and nitrogen atoms are red and blue, respectively. (b) AUC analysis of the head region alone, FliD_78–273, at pH 8.0 (upper panel) and pH 5.0 (lower panel) reveals a monomeric species at pH 8.0 and the additional minor presence of a dimeric species at pH 5.0. (c) Silver-stained SDS-PAGE after chemical crosslinking of FliD_78–273. (d) SAXS analysis of FliD_78–273. 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 FliD_7–273 at 1.34 mg/mL (blue), 0.67 mg/mL (red, used to calculate the envelope) and 0.335 mg/mL (green). (e) Silver-stained SDS-PAGE after chemical crosslinking of FliD_78–474. (f) Silver-stained SDS-PAGE after chemical crosslinking of FliD_1–405. DOI: http://dx.doi.org/10.7554/eLife.18857.013

Figure 6.. Small angle X-ray scattering (SAXS) data of FliD_1–405, FliD_78–474 and FliD_1–474.

Log-scale intensity SAXS profiles, Kratky Plot (I × q² versus q), radial distribution function calculated by GNOM and SAXS envelopes calculated by DAMMIF are shown for: (a) FliD_1–405 at 10.4 mg/mL (blue), 5.2 mg/mL (red) and 2.6 mg/mL (grey); (b) FliD_78–474 at 9.9 mg/mL (blue), 4.95 mg/mL (red) and 2.5 mg/mL (grey); and (c) FliD_1–474 at 11 mg/mL (blue), 5.5 mg/mL (red), 2.7 mg/mL (grey), 1.38 mg/mL (green) and 0.69 mg/mL (yellow). DOI: http://dx.doi.org/10.7554/eLife.18857.014

Figure 6—figure supplement 1.. Analytical ultracentrifugation (AUC) analysis of FliD_1–474 at pH 11.0.

AUC analysis of FliD_1–474 at pH 11.0 indicates that the protein is monomeric at pH 11.0. DOI: http://dx.doi.org/10.7554/eLife.18857.015

Figure 7.. Regions of Pseudomonas FliD outside of the head domains and initial leg helix are highly dynamic.

(a) Hydrogen/deuterium exchange analysis of FliD_78–405. Percent deuteration (%D) heat map is shown. Peptides exhibiting EX1 kinetics are indicated. (b) Mass spectra of four FliD peptides exhibit double isotopic envelopes characteristic of EX1 kinetics (below). Three of these peptides are mapped to the crystal structure (above; FliD hexamers are in green and gold). (c) Conformational stability as determined by hydrogen/deuterium exchange mapped to the crystal structure of FliD_78–405 using the same color coding for %D as shown in (a). DOI: http://dx.doi.org/10.7554/eLife.18857.016

Figure 7—figure supplement 1.. Circular dichroism analysis of FliD variants.

(a) CD spectra recorded from 190 to 260 nm and (b) melting curves recorded from 15 to 90°C are shown for FliD_78–405, FliD_1–474, FliD_78–273, FliD_78–474 and FliD_1–405. Melting curves were recorded at 222 nm for all proteins except for FliD_78–273, which was analyzed at 205 nm. DOI: http://dx.doi.org/10.7554/eLife.18857.017

Figure 8.. Interaction of Pseudomonas FliD regions.

(a) Difference plot of hydrogen/deuterium exchange data from full length FliD_1–474 and the crystallized fragment, FliD_78–405. (b) Hydrogen/deuterium exchange for peptides corresponding to residues 166–176 (top, marked by + in (a) and (b)) and residues 225–239 (bottom, marked by * in (a) and (b)). Positions of peptides 166–176 and 225–239 in the FliD_78–405 crystal structure (right). (c) Difference plots of hydrogen/deuterium exchange data from full length FliD_1–474 and the fragments missing only the C-terminal coiled coil, FliD_1–405, only the N-terminal coiled coil, FliD_78–474, or both the N- and C-terminal coiled coils and the leg domain, FliD_78–273 (top, middle and bottom, respectively). (d) Schematic model of the FliD monomeric subunit showing the N-terminal coiled coil stabilizing the head 1 and foot domains and also interacting with the C-terminal coiled coil. DOI: http://dx.doi.org/10.7554/eLife.18857.018

Figure 8—figure supplement 1.. Hydrogen-deuterium exchange-mass spectrometry analysis of FliD variants.

Heat maps of hydrogen/deuterium exchange for FliD_1–474, FliD_78–405, FliD_78–474, FliD_1–405 and FliD_78–273, from top to bottom, respectively. DOI: http://dx.doi.org/10.7554/eLife.18857.019

Figure 8—figure supplement 2.. Analytical ultracentrifugation (AUC) analysis of FliD_1–474 at 4 μM.

AUC analysis of FliD_1–474 at a concentration of 4 μM at pH 8.0 indicates that the protein is predominantly monomeric in an equilibrium with dimeric species at 4 μM and pH 8.0. DOI: http://dx.doi.org/10.7554/eLife.18857.020

Figure 8—figure supplement 3.. FliD intrinsic disorder analysis.

Intrinsic disorder prediction of FliD_1–474 using publicly available algorithms (as indicated, left column). Averaging of the results (color, bottom row) was done giving all servers equal weight. DOI: http://dx.doi.org/10.7554/eLife.18857.021