Research ArticleBIOMOLECULES

Evolutionary fine-tuning of conformational ensembles in FimH during host-pathogen interactions

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Science Advances  10 Feb 2017:
Vol. 3, no. 2, e1601944
DOI: 10.1126/sciadv.1601944
  • Fig. 1 Structure-function relationship in the type 1 adhesin FimH.

    (A) Schematic representation of FimH sequence. The lectin domain (FimHLD, residues 1 to 155) is colored blue, the linker is colored yellow (residues 156 to 160), and the pilin domain (FimHPD, residues 161 to 279) is colored teal. (B) Comparison of previously identified conformations of FimH. FimH bound to FimC in a FimCH complex exists in a high-affinity conformer, or R state, with an elongated orientation between FimHLD and FimHPD, a narrowly packed β-sandwich fold in FimHLD (as highlighted between the two black triangles), and packed mannose-binding loops (labeled as L1, L2, and L3). FimH in a tip assembly (FimCFFGH complex) adopts a low-affinity conformer, or T state, with a compacted orientation between FimHLD and FimHPD, a widened β-sandwich fold in FimHLD (as highlighted between the two black triangles), and displaced binding loops (particularly L1). Positively selected residues are indicated as red spheres, and the mannose-binding pocket is shaded gray. (C) Schematic representation of the negatively coupled allosteric relationship between mannose and the interface between FimH domains, whereby increases in mannose binding disfavor contacts between FimHLD and FimHPD and vice versa.

  • Fig. 2 Crystal structures and mannose binding of FimH variants in a tip-like setting.

    (A) Reaction scheme of in vitro DSE reaction to produce tip-like FimGNteH complexes. (B) Representative SDS–polyacrylamide gel electrophoresis of purified FimGNteH variants either boiled (not labeled) or not boiled (NB). (C) Enzyme-linked immunosorbent assay (ELISA) measuring binding of FimGNteH variant complexes to surface-coated glycoproteins, which include secretory IgA (sIgA), Tamm-Horsfall protein (THP), collagen IV, and laminin. (D) Crystal structures of FimGNteH A62S [Protein Data Bank (PDB) ID 5JQI, left] and FimGNteH A27V/V163A (PDB ID 5JR4, right) depicted as ribbons. These structures are overlaid on previously solved crystal structures of FimH in a FimCFFGH complex (3JWN) and FimCH complex (1KLF), respectively. Conformations are labeled accordingly. FimHLD, linker, and FimHPD are colored as in (A), the insertion loop (residues 109 to 124) is colored purple, and FimGNte is colored gray. (E) FimHLD-FimHPD interface in FimGNteH A62S (left) and FimGNteH A27V/V163A (right). Contacts between residues are indicated as black dotted lines. (F) Structural alignment of FimGNteH A27V/V163A (colored blue) to FimHLD of mannose-bound FimCH (colored white). Residue side chains and mannose in green are depicted as sticks. Contacts between mannose and FimH are indicated as black dotted lines.

  • Fig. 3 Conformational ensembles of apo and ligated FimH variants in solution.

    (A) Structural comparison heat map of SAXS profiles indicates varying degrees of conformational similarity among FimGNteH variants, as measured by χ2, ranging from high (blue) to low (red) similarity. (B) Normalized pair distance distributions of FimGNteH variants in the absence or presence of 4Z269. FimGNteH variants are represented as solid lines in the absence of 4Z269 or dotted lines in the presence of 4Z269 and color-coded, as indicated by the colored lines in (A). (C) Structural comparison heat map indicates varying degrees of conformational similarity of each FimGNteH variant in the absence or presence of mannoside 4Z269 at a 2× molar ratio. Color-coded as in (A). (D) Averaged ab initio models of FimGNteH variants in the absence or presence of 4Z269 are color-coded, as previously indicated.

  • Fig. 4 Conformational distributions of free and 4Z269-bound FimGNteH variants isolated in the gas phase, as revealed by IMMS.

    (A) CCS distributions of intact FimGNteH variant complexes measured by IMMS. (B) Comparison of CCS distributions of free (solid line) and 4Z269-bound (dotted line) FimGNteH variants. The solid and dotted black lines represent fitted Gaussian distributions to apo and ligated FimGNteH, respectively. Fitted Gaussian distributions are labeled by letters, given their mean CCS values. Note that Q133K cannot bind mannose and that the dotted lines for this variant represent CCS distributions and Gaussian fits to an independently measured apo FimGNteH Q133K spectral peak from the sample that was treated with 4Z269.

  • Fig. 5 Dynamics and binding mechanisms of conformational populations in FimGNteH WT.

    (A) Structures revealed by MD simulations of FimGNteH WT in a T conformation (top) or bent R conformation (bottom) with corresponding measures of structural fluctuation over time (RMSD) and distributions of sampled protein shapes (Rg). Different colors correspond to four independent simulation replicates. (B) Three-dimensional conformational phase space of FimGNteH as defined by bend, twist, and orientation angles for simulations initiated from the T (green), bent R (blue), or elongated R (cyan) conformation. Shadows are cast on the grid panels and colored in gray. (C) Structures revealed by MD simulations of FimGNteH WT in a T conformation (top) or bent R conformation (bottom) in the presence of mannose, with corresponding measures of structural fluctuation over time (RMSD), distributions of sampled protein shapes (Rg), and mannose binding. “On” and “Off” measure whether the center of mass of mannose is within or outside 10 Å of the carbonyl of residue F1 in the binding pocket. (D) Representative binding modes of mannose for T (top) and bent R (bottom) after 5 ns. Mannose is depicted as sticks, whereas FimH is shown as a ribbon representation. (E) Structures revealed by MD simulations of FimGNteH WT in a T (top) or bent R (bottom) conformation in the presence of oligomannose-3 (Man(α1–3)-[Man(α1–6)]-Man), with corresponding measures of structural fluctuation over time (RMSD), distributions of sampled protein shapes (Rg), and mannose binding. “On” and “Off” measure whether the center of mass of oligomannose-3 is within or outside 20 Å of the carbonyl of residue F1 in the binding pocket. (F) Representative binding modes of oligomannose-3 for T (top) and bent R (bottom) after 8.5 ns. Man(α1–3)-[Man(α1–6)]-Man is depicted as sticks and colored green, yellow, and cyan, respectively.

  • Fig. 6 Role of FimH conformation in bladder colonization during UTI.

    (A) Bacterial titers of mouse bladders infected with UTI89 harboring either FimH WT (blue) or A27V/V163A (orange) at an inoculum of 107 colony-forming units (CFU) measured at 1, 3, and 6 hpi. (B) Total bacterial titers of 5637 bladder epithelial cells (no gentamicin treatment) infected with UTI89 harboring WT (blue), Q133K (red), A62S (green), or A27V/V163A (orange) FimH at an inoculum of 107 CFU. (C) Invaded bacterial titers of 5637 bladder epithelial cells (treated with gentamicin) infected with UTI89 harboring WT (blue), Q133K (red), A62S (green), or A27V/V163A (orange) FimH at an inoculum of 107 CFU. LOD, limit of detection. (D and E) Bacterial titers of C57BL/6 mouse bladders without catheterization or bladders and implants infected with UTI89 harboring either FimH WT (blue) or A27V/V163A (orange) at an inoculum of 107 CFU 24 hours after catheterization. **P < 0.01, ***P < 0.001, ****P < 0.0001, two-tailed Mann-Whitney U test.

  • Fig. 7 Proposed model of FimH conformational ensembles, mannose binding, and virulence in UTI.

    (A) Two-state conformational landscape of FimH. FimH at the pilus tip natively adopts an equilibrium of a single, dynamically restrained, low-affinity T state and multiple, highly dynamic, high-affinity R states with various bends, twists, and orientations. Positively selected residues can shift this preexisting conformational equilibrium and thereby influence mannose-binding affinity. The T and R states can bind mannose. Mannose in a tilted orientation rapidly enters into the widened and shallow binding pocket of the T state. Mannosylated ligands in a bound T state can then rotate in a high-affinity orientation and allosterically trigger structural perturbations that disrupt FimHLD and FimHPD interactions and facilitate conversion to the bound R state. In addition, mannose in a horizontal orientation can less rapidly engage the R state but does so very tightly through hydrogen bond interactions with several binding loop residues. Positive selection, in modulating a native conformational equilibrium, likely alters flux through these two distinct binding mechanisms. (B) Schematic model of the FimH molecular tether. The bends, twists, and orientations between FimHLD and FimHPD adopted in bound R states argue for a model in which the pilus tip can bend and rotate at the site of the FimH linker with an immobilized, bound FimHLD. This physical tethering in theory increases the biophysical and functional adaptability of the pilus and thereby allows bacteria to remain attached to the bladder epithelium. (C) Pathogenesis outcomes depend on the preexisting equilibrium and affinity of FimH, whereby moderate affinity is ideal for successful colonization of the bladder epithelium and formation of IBCs. Catheterization allows the high-affinity variant A27V/V163A to partially circumvent the colonization resistance property observed in the intact, unperturbed bladder habitat.

Supplementary Materials

  • Supplementary material for this article is available at http://advances.sciencemag.org/cgi/content/full/3/2/e1601944/DC1

    fig. S1. Structural analysis of solved FimGNteH complex crystal structures.

    fig. S2. Structural comparison of all known FimH conformations.

    fig. S3. Solution analysis of FimCH and FimGNteH variants.

    fig. S4. Native and ion mobility mass spectra of FimGNteH variants in the absence or presence of 4Z269.

    fig. S5. Conformational dynamics and binding mechanisms of FimGNteH WT.

    table S1. Data collection and refinement statistics.

    table S2. Fitted parameter values and analysis of Gaussian peaks on CCS distributions.

    movie S1. Combined MD simulation trajectories initiated from the T state in the absence of mannose.

    movie S2. Combined MD simulation trajectories initiated from the bent R state in the absence of mannose.

    movie S3. Combined MD simulation trajectories initiated from the elongated R state in the absence of mannose.

    movie S4. Combined MD simulation trajectories initiated from the T state in the presence of mannose.

    movie S5. Combined MD simulation trajectories initiated from the bent R state in the presence of mannose.

    movie S6. Combined MD simulation trajectories initiated from the elongated R state in the presence of mannose.

    movie S7. Combined MD simulation trajectories initiated from the T state in the presence of oligomannose-3.

    movie S8. Combined MD simulation trajectories initiated from the bent R state in the presence of oligomannose-3.

  • Supplementary Materials

    This PDF file includes:

    • fig. S1. Structural analysis of solved FimGNteH complex crystal structures.
    • fig. S2. Structural comparison of all known FimH conformations.
    • fig. S3. Solution analysis of FimCH and FimGNteH variants.
    • fig. S4. Native and ion mobility mass spectra of FimGNteH variants in the absence or presence of 4Z269.
    • fig. S5. Conformational dynamics and binding mechanisms of FimGNteH WT.
    • table S1. Data collection and refinement statistics.
    • table S2. Fitted parameter values and analysis of Gaussian peaks on CCS distributions.
    • Legends for movies S1 to S8

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    Other Supplementary Material for this manuscript includes the following:

    • movie S1 (.mpg format). Combined MD simulation trajectories initiated from the T state in the absence of mannose.
    • movie S2 (.mpg format). Combined MD simulation trajectories initiated from the bent R state in the absence of mannose.
    • movie S3 (.mpg format). Combined MD simulation trajectories initiated from the elongated R state in the absence of mannose.
    • movie S4 (.mpg format). Combined MD simulation trajectories initiated from the T state in the presence of mannose.
    • movie S5 (.mpg format). Combined MD simulation trajectories initiated from the bent R state in the presence of mannose.
    • movie S6 (.mpg format). Combined MD simulation trajectories initiated from the elongated R state in the presence of mannose.
    • movie S7 (.mpg format). Combined MD simulation trajectories initiated from the T state in the presence of oligomannose-3.
    • movie S8 (.mpg format). Combined MD simulation trajectories initiated from the bent R state in the presence of oligomannose-3.

    Download Movies S1 to S8

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