Structural Basis for Sialoglycan Binding by the Streptococcus sanguinis SrpA Adhesin

Abstract

Streptococcus sanguinisis a leading cause of infective endocarditis, a life-threatening infection of the cardiovascular system. An important interaction in the pathogenesis of infective endocarditis is attachment of the organisms to host platelets.S. sanguinisexpresses a serine-rich repeat adhesin, SrpA, similar in sequence to platelet-binding adhesins associated with increased virulence in this disease. In this study, we determined the first crystal structure of the putative binding region of SrpA (SrpABR) both unliganded and in complex with a synthetic disaccharide ligand at 1.8 and 2.0 Å resolution, respectively. We identified a conserved Thr-Arg motif that orients the sialic acid moiety and is required for binding to platelet monolayers. Furthermore, we propose that sequence insertions in closely related family members contribute to the modulation of structural and functional properties, including the quaternary structure, the tertiary structure, and the ligand-binding site.

Keywords: Streptococcus; adhesin; bacterial adhesion; bacterial pathogenesis; carbohydrate-binding protein; crystal structure; immunoglobulin-like domain; lectin; sialic acid.

FIGURE 1.

Structure of the SrpA_BR protomer. Ribbons diagram of SrpA_BR protomer colored by sequence with the N terminus in blue and the C terminus in red. Assigned Ca²⁺ ions are shown as yellow spheres.

FIGURE 2.

Structure-based sequence alignment of SrpA and GspB. A, sequence alignment was performed manually by overlaying the structures of SrpA_BR and GspB_BR. Secondary structural elements are shown above and below the sequence. Amino acids in red text are identical. The Thr-Arg motif is highlighted in green. The insertions in GspB highlighted in yellow are near the ligand binding pocket, with residues 449–451 forming a short loop and residues 498–514 forming the helix at the carbohydrate binding pocket. Insertions in SrpA highlighted in purple are predicted to influence the interdomain angle, and the insertion in GspB highlighted in red is predicted to prevent dimerization. B, ribbon diagram of SrpA_BR with sequence inserts and binding site residues colored according to the sequence alignment and superimposed onto a surface where the Siglec domain is brown and the Unique domain is blue. C, ribbon diagram of the GspB_BR Siglec and Unique domains with sequence inserts and binding site residues colored according to the sequence alignment and superimposed onto a surface of the binding region.

FIGURE 3.

Structure of the SrpA_BR dimer. A, ribbon diagram of the crystallographic asymmetric unit containing the SrpA_BR dimer colored by domain. The N-terminal Siglec domains are shown in brown, and the C-terminal Unique domains are shown in blue. Electron density assigned as Ca²⁺ ions are shown as yellow spheres. The view is down the non-crystallographic 2-fold axis of symmetry. B, view of the Ca²⁺ ion mediating dimerization between the Unique domains. Side chains involved in a hydrogen-bonding network that contributes to Ca²⁺ coordination are highlighted. The view is the same as in A. C, modeling of the GspBUnique domain (gray) into the same orientation as the SrpA Unique domain dimer identifies a sequence insertion of seven amino acids (residues 564–571; red) that sterically prevents oligomerization in the same manner as is observed in the SrpA_BR crystal structure.

FIGURE 4.

Domain orientation of the SrpA_BR protomer comparison of the SrpA_BR Siglec and Unique domains (brown and blue) to the GspB_BR domains (gray) highlights the difference in interdomain angle between the two structures. Two sequence insertions in SrpA (residues 335–342 and residues 373–376; magenta) as compared with GspB prevent these two homologs from adopting the same interdomain orientation. Ca²⁺ ions are omitted for clarity.

FIGURE 5.

Carbohydrate binding in SrpA_BR. A and B are superimposed with |F_o| − |F_c| difference electron density contoured at 3σ and calculated after removal of the ligand and refinement in Phenix. A, stereoview of the sialyl galactoside disaccharide bound to SrpA_BR highlights the quality of the electron density map. B, surface representation of SrpA_BR focusing on one protomer of the dimer. The Siglec domain is colored in brown, and the Unique domain is colored in blue. The view highlights large and open binding pocket for carbohydrate ligand. C, surface representation of GspB_BR with the same orientation of the carbohydrate-binding Siglec domain as SrpA_BR in B. The sequence inserts of GspB_BR with respect to SrpA_BR are colored using the same coloring scheme as is used in Fig. 2. To mark the global location of the ligand binding pocket, a Neu5Acα2–3Gal disaccharide is modeled using the position from SrpA_BR; however, its position is not experimentally determined.

FIGURE 6.

Role of the Thr-Arg motif in orienting carbohydrate ligand. A and B, zoomed in view of the Neu5Gcα2–3GalβOMe sialyl galactoside disaccharide-binding site of SrpA_BR (shown as sticks) in the context of the binding pocket (shown as a surface). A, SrpA_BR, the disaccharide position is experimentally determined; B, GspB_BR, the disaccharide is modeled based upon the Thr-Arg motif and is not experimentally determined. The view highlights the conservation of the hydrogen bond locations provided by the Thr-Arg motif and the more restricted binding pocket of GspB_BR that likely restricts the range of carbohydrates that can bind. C, stereoview of sialyl galactoside in the binding site of SrpA_BR. Distances consistent with hydrogen bonding interactions are shown as dotted lines.

FIGURE 7.

Validation of the ligand binding pocket by mutagenesis. A, binding of wild-type and variant SrpA to platelet monolayers. ULI indicates the inclusion of seven amino acids of the GspB Unique domain into the equivalent position on the SrpA Unique domain. B, overlay of the Siglec domain of the disaccharide-bound wild-type SrpA_BR (gray) with regions of the SrpA_BR R347E variant that exhibited conformational difference (teal). The comparison identifies significant differences of positions between the two loops that sterically occlude the sialic acid binding pocket in the R347E variant. The location of the Cα atom of Arg-347 is indicated with a cyan sphere.