The Streptococcus gordonii Adhesin CshA Protein Binds Host Fibronectin via a Catch-Clamp Mechanism

Abstract

Adherence of bacteria to biotic or abiotic surfaces is a prerequisite for host colonization and represents an important step in microbial pathogenicity. This attachment is facilitated by bacterial adhesins at the cell surface. Because of their size and often elaborate multidomain architectures, these polypeptides represent challenging targets for detailed structural and functional characterization. The multifunctional fibrillar adhesin CshA, which mediates binding to both host molecules and other microorganisms, is an important determinant of colonization by Streptococcus gordonii, an oral commensal and opportunistic pathogen of animals and humans. CshA binds the high-molecular-weight glycoprotein fibronectin (Fn) via an N-terminal non-repetitive region, and this protein-protein interaction has been proposed to promote S. gordonii colonization at multiple sites within the host. However, the molecular details of how these two proteins interact have yet to be established. Here we present a structural description of the Fn binding N-terminal region of CshA, derived from a combination of X-ray crystallography, small angle X-ray scattering, and complementary biophysical methods. In vitro binding studies support a previously unreported two-state "catch-clamp" mechanism of Fn binding by CshA, in which the disordered N-terminal domain of CshA acts to "catch" Fn, via formation of a rapidly assembled but also readily dissociable pre-complex, enabling its neighboring ligand binding domain to tightly clamp the two polypeptides together. This study presents a new paradigm for target binding by a bacterial adhesin, the identification of which will inform future efforts toward the development of anti-adhesive agents that target S. gordonii and related streptococci.

Keywords: X-ray crystallography; adhesin; bacterial pathogenesis; intrinsically disordered protein; microbiology; small-angle X-ray scattering (SAXS).

FIGURE 1.

Reassigned domain architecture of CshA. The CshA polypeptide comprises an N-terminal leader peptide (residues 1–40), a three-domain non-repetitive region (residues 41–819), a repeat region formed from 17 90–102-aa domains (residues 820–2507), and a C-terminal LPXTG cell wall anchor motif. The CshA domains NR1, NR2, NR3, and R13, produced recombinantly during this study, are highlighted (masses include vector encoded His₆-tag and linker).

FIGURE 2.

CshA-dependent adherence of S. gordonii and L. lactis strains to immobilized cellular fibronectin. A, dot immunoblot analysis of CshA expression levels on the surface of S. gordonii and L. lactis strains. B, comparative levels of cFn adhesion by S. gordonii and L. lactis strains expressing CshA proteins. Mean values from four independent experiments are shown. Error bars represent standard deviations from the mean. *, p < 0.05; **, p < 0.006 as established by analysis of variance using Dunnett's multiple comparison test. ns, not significant.

FIGURE 3.

Sensorgrams of fibronectin binding by CshA recombinant domains and control proteins. Representative of the binding of each CshA fragment (NR1, NR2, NR3, and R13), BSA and Fn antibody, to either plasma (A) or cellular (B) Fn. Note: the K_D values are computationally calculated based on the exact amount of Fn deposited on the biosensor tip, which varies from run to run.

FIGURE 4.

Biophysical and structural characterization of CshA NR1. A, PONDR-FIT analysis of the CshA NR1 aa sequence. B, SDS-PAGE analysis of recombinant NR1. The predicted molecular mass of the protein based on amino acid composition is 20.8 kDa. C, far-UV CD spectrum of CshA_NR1. The trace shown is a mean average calculated from 10 scans of the same protein sample. D, SAXS scattering profile of CshA NR1. A Kratky plot derived from the scattering profile is shown as an inset.

FIGURE 5.

Structural studies of CshA NR2. A, X-ray crystal structure of the CshA NR2 strand-swapped dimer. Individual monomers are colored yellow and purple, respectively. The location of loop regions containing residues 313–331 and 393–397 for which convincing electron destiny was not observed are indicated by dashed lines. B, structural model of CshA NR2 based on the strand-swapped dimer crystal structure. The location of the ligand-binding site and the associated capping loop are indicated. C, details of the CshA NR2 ligand-binding site, including electrostatic charge distribution (blue, positive; red, negative) and aa composition.

FIGURE 6.

Comparison of CshA NR2 with structurally related homologues. Structures of the N-terminal domain of S. gordonii Sgo0707 (green), the AgI/II polypeptide V domains of Streptococcus mutans SpaP (orange) and S. gordonii SspB (blue), CshA_NR2 (model based on the strand-swapped dimer crystal structure; cyan), and T. maritima TmCBM61 (yellow) are shown. The conserved β-sandwich core of each protein is highlighted with a blue rectangle.

FIGURE 7.

Schematic depicting the catch-clamp mechanism of Fn binding by CshA. The intrinsically disordered NR1 domain of the protein rapidly engages and binds Fn in a process expedited by the sizable capture radius of the domain. Fn binding results in the formation of a dissociable pre-complex that may be accompanied by the recovery of NR1 secondary structure. The resulting pre-complex is stabilized by a high affinity binding interaction mediated by the NR2 domain of CshA.