A structural model for binding of the serine-rich repeat adhesin GspB to host carbohydrate receptors

Abstract

GspB is a serine-rich repeat (SRR) adhesin of Streptococcus gordonii that mediates binding of this organism to human platelets via its interaction with sialyl-T antigen on the receptor GPIbα. This interaction appears to be a major virulence determinant in the pathogenesis of infective endocarditis. To address the mechanism by which GspB recognizes its carbohydrate ligand, we determined the high-resolution x-ray crystal structure of the GspB binding region (GspB(BR)), both alone and in complex with a disaccharide precursor to sialyl-T antigen. Analysis of the GspB(BR) structure revealed that it is comprised of three independently folded subdomains or modules: 1) an Ig-fold resembling a CnaA domain from prokaryotic pathogens; 2) a second Ig-fold resembling the binding region of mammalian Siglecs; 3) a subdomain of unique fold. The disaccharide was found to bind in a pocket within the Siglec subdomain, but at a site distinct from that observed in mammalian Siglecs. Confirming the biological relevance of this binding pocket, we produced three isogenic variants of S. gordonii, each containing a single point mutation of a residue lining this binding pocket. These variants have reduced binding to carbohydrates of GPIbα. Further examination of purified GspB(BR)-R484E showed reduced binding to sialyl-T antigen while S. gordonii harboring this mutation did not efficiently bind platelets and showed a significant reduction in virulence, as measured by an animal model of endocarditis. Analysis of other SRR proteins revealed that the predicted binding regions of these adhesins also had a modular organization, with those known to bind carbohydrate receptors having modules homologous to the Siglec and Unique subdomains of GspB(BR). This suggests that the binding specificity of the SRR family of adhesins is determined by the type and organization of discrete modules within the binding domains, which may affect the tropism of organisms for different tissues.

Figure 1. Schematic of the SRR adhesins.

Selected SRR adhesins are aligned based on the N-terminus of their binding regions, with the largest binding region at the top and the smallest binding region at the bottom. The length of each domain and the entire protein is drawn to-scale. SP = signal peptide (dark gray), SRR1 = first serine rich repeat (light gray), BR = binding region (red), SRR2 = second serine rich repeat (dark gray), CWA = cell wall anchoring motif (light gray). The SRR adhesins used in this figure are S. aureus SraP (SraP); S. epidermidis SRR1 (seSRR), S. agalactiae SRR1 (SRR1_GBS), S. gordonii strain M99 GspB (GspB), S. parasanguinis Fap1 (Fap1), S. pneumoniae PsrP (PsrP), S. gordonii strain Challis Hsa (Hsa), and S. sanguinis SrpA (SrpA).

Figure 2. Structure of GspB_BR.

A. Two views of the structure of GspB_BR separated by a 90° rotation. The N-terminus of the binding region is located at the top of the figure and the C-terminus is located at the bottom. β-Strands are colored magenta, α-helices are colored blue, and loops are colored black. A cation bound within the Siglec domain is shown as a purple sphere. B. Representative experimental electron density (blue mesh) at 2.0 Å resolution, contoured at 1.0 σ, and depicted superpositioned onto residues 515–520 of the final model. C. A m|F_o|−d|F_c| omit electron density map at 1.4 Å resolution (green mesh) calculated in REFMAC5 after the removal of the model contoured at 3.0 σ and depicted superpositioned onto residues 515–520 of the final model. D. Folding diagram of the Unique subdomain. β-Strands are colored magenta and the α-helix is colored blue.

Figure 3. Ig-fold topologies.

A. Topology diagram of a C-set Ig-fold. B. Topology diagram of the DE-variant of a C-set Ig-fold identified in MSCRAMMs . C. Topology diagram of the GspB_BR CnaA subdomain. D. Topology diagram of a V-set Ig-fold. E. Topology diagram of a Siglec showing the binding location of the sialylated carbohydrate in pink. F. Topology diagram of the GspB_BR Siglec subdomain showing the binding location of the sialyl-T antigen in pink.

Figure 4. Identification of the receptor binding site.

A. Inhibition of S. gordonii strain M99 binding to immobilized glycocalicin by the α-2,3-sialyl (1-thioethyl)galactose disaccharide. Binding was assessed in the absence or presence of 44 mM disaccharide, and is expressed as the percent of input bacteria that remained bound to glycocalicin after repeated washing of the microtiter wells (mean ± standard deviation). B. Stereo view of the disaccharide binding site superpositioned with m|F_o|−d|F_c| omit electron density (green mesh) calculated in REFMAC5 and contoured to 2.5 σ after the removal of disaccharide from the model. C. A space filling model of GspB_BR with bound disaccharide (stick model) demonstrates that the carbohydrate receptor binds within a pocket on the surface of the protein. The protein surface is colored by subdomain with the Siglec subdomain in yellow and the Unique subdomain in blue. The stick model of the disaccharide has carbon atoms colored magenta, oxygen atoms colored red, the nitrogen atom colored blue, and the sulfur atom colored yellow. The model is rotated 50° along the y-axis and 40° along the x-axis as compared to panel B. D. Overview of the GspB_BR structure highlighting the location of the disaccharide binding site in the Siglec subdomain. The model is rotated 90° along the x-axis as compared to panel B. The disaccharide is shown with magenta bonds and the cation is highlighted as a purple sphere.

Figure 5. Comparison of carbohydrate binding in the Siglec subdomain of GspB_BR and Siglec-5.

A. Overlay of the Siglec subdomain of GspB_BR and Siglec-5. Siglec-5 is colored teal with the carbons of 3′ sialyllactose colored purple. The Siglec subdomain of GspB_BR is colored yellow with the carbons of α-2,3-sialyl (1-thioethyl)galactose colored in magenta. In this panel, the Siglec carbohydrate binding site is occupied by a helix in the GspB_BR Siglec subdomain therefore eliminating the possibility that this could be a second binding site for GspB_BR. B & C. Comparison of the location and details of the carbohydrate binding site in (B) GspB_BR with that of (C) Siglec-5 (PDB entry 2ZG3; [45]). The Siglec subdomain of GspB_BR is colored yellow with the carbons of α-2,3-sialyl (1-thioethyl)galactose colored in magenta and Siglec-5 is colored teal with the carbons of 3′ sialyllactose colored purple. While Siglec-5 is used as an example, it is noted that for all structurally characterized Siglecs (Siglec-1 (aka sialoadhesin); PDB entry 1QFO; [44]), Siglec-5 (2ZG3; [45]), and Siglec-7 (PDB entry 2HRL; [74])), the sialic acid binding pocket is found at the same location.

Figure 6. Interdomain Angle.

Differences in interdomain angles between structures of GspB_BR. The structures of the Siglec and Unique subdomains of unbound and disaccharide-bound GspB_BR were aligned using Dyndom . The unbound structure is colored in silver, and the structure in complex with α-2,3-sialyl (1-thioethyl) galactose is colored by subdomain with the CnaA subdomain in red, the Siglec subdomain in yellow and the Unique subdomain colored in blue, and cation colored purple. The side chains of hinge residues Lys398, Asp399, and Thr400 are highlighted in the boxed inset. The difference in interdomain angle between the two structures is a 40°. A movie morphing these states is seen in Video S1 .

Figure 7. Amino acid substitutions in the carbohydrate binding site of GspB_BR.

A. Expression of GspB and variant proteins on the S. gordonii cell surface. Peptidoglycan-linked proteins were extracted from the bacterial cell surface and analyzed by western blotting using a polyclonal anti-GspB serum (upper panel). The nitrocellulose membrane was then re-probed using a polyclonal anti-GspA serum (lower panel) in order to assess the relative efficiency of protein extraction and protein loading (GspA is another LPXTG-linked protein that is expressed by S. gordonii M99). B. Binding of S. gordonii strain M99 and derivative strains to glycocalicin immobilized in microtiter wells. Binding is expressed as the percent of input bacteria that remained bound to glycocalicin after repeated washing of the wells (mean ± standard deviation). The labels of each column indicate the sequence of the protein expressed in S. gordonii, with M99 indicating wild type, ΔgspB indicating a strain where the gene encoding GspB has been deleted, and remaining columns indicating the amino acid substitution present in the protein. E401 is a control residue not located near the binding pocket. C. Binding of sialyl-T antigen to purified GST-GspB_BR. The indicated amounts of GST or GST-GspB_BR wild-type (wt) and variant (R484E) proteins were immobilized in microtiter wells, and the binding of biotinylated sialyl-T antigen to each protein was detected by using peroxidase-conjugated streptavidin along with a chromogenic peroxidase substrate. Binding is expressed as the mean ± standard deviation (n = 3). The o represents purified GST-GspB_BR-R484E, the x represents purified GST, and the represents purified GST-GspB_BR. D. Binding of M99 and derivative strains to human platelets. Binding is expressed as the percent of input bacteria that remained bound to platelets after repeated washing of the wells (mean ± standard deviation).

Figure 8. Binding of wild type S. gordonii strain M99, PS2116 (strain M99 with gspb harboring the R484E substitution), and PS846 (ΔgspB) to platelets.

Fixed platelets were adhered to poly-L-lysine coated cover slips, quenched, and bound to live bacteria stained with DAPI. A–C. Fluorescent images of each strain with DAPI stain. D–F. Merge between the DIC image and the DAPI image to show relative number of platelets.

Figure 9. Modular organization of the binding regions within adhesins of the SRR superfamily.

A. A summary of BLAST or ClustalW sequence alignments of the binding regions of SRR adhesins. The first number indicates sequence identity and the second indicates sequence similarity. GspB_BR has been divided into its subdomains, with GspB_BR-C indicating the CnaA subdomain and GspB_BR-SU indicating both the Siglec and Unique subdomains. Only the C-terminal subdomain of SRR1_GBS (SRR1_GBS-β) and Fap1_NR (Fap1_NR-β) are used in this analysis since the N-terminal domains do not share detectable sequence identity with other sequences used in this analysis. Boxes in grey indicate SRR pairs that do not have detectable sequence identity within the binding region. B. A schematic summarizing (A) The colored rectangles represent similar regions of sequence, and are drawn to scale. If known, representative structures of each domain are illustrated along the top, and binding partners are indicated along the side.