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• W2272772088 abstract "Streptococcus sanguinis is 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. sanguinis expresses 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. Streptococcus sanguinis is 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. sanguinis expresses 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. Infective endocarditis is associated with significant morbidity and mortality (1.Hoen B. Duval X. Clinical practice. Infective endocarditis.N. Engl. J. Med. 2013; 368: 1425-1433Crossref PubMed Scopus (345) Google Scholar). Although adherence between pathogen and host platelets is one of the first required steps for infection (2.Fitzgerald J.R. Foster T.J. Cox D. The interaction of bacterial pathogens with platelets.Nat. Rev. Microbiol. 2006; 4: 445-457Crossref PubMed Scopus (351) Google Scholar, 3.Sullam P.M. Sande M.A. Role of platelets in endocarditis: clues from von Willebrand disease.J. Lab. Clin. Med. 1992; 120: 507-509PubMed Google Scholar), the pathogenesis of infective endocarditis is a complex process that is not well understood. The viridans group of streptococci, including Streptococcus sanguinis and Streptococcus gordonii, accounts for an estimated 17–45% of all cases of infective endocarditis (4.Murdoch D.R. Corey G.R. Hoen B. Miró J.M. Fowler Jr., V.G. Bayer A.S. Karchmer A.W. Olaison L. Pappas P.A. Moreillon P. Chambers S.T. Chu V.H. Falcó V. Holland D.J. Jones P. et al.Clinical presentation, etiology, and outcome of infective endocarditis in the 21st century: the International Collaboration on Endocarditis-Prospective Cohort Study.Arch. Intern. Med. 2009; 169: 463-473Crossref PubMed Scopus (1480) Google Scholar, 5.Thornhill M.H. Dayer M.J. Forde J.M. Corey G.R. Chu V.H. Couper D.J. Lockhart P.B. Impact of the NICE guideline recommending cessation of antibiotic prophylaxis for prevention of infective endocarditis: before and after study.BMJ. 2011; 342: d2392Crossref PubMed Scopus (193) Google Scholar, 6.Tleyjeh I.M. Steckelberg J.M. Murad H.S. Anavekar N.S. Ghomrawi H.M. Mirzoyev Z. Moustafa S.E. Hoskin T.L. Mandrekar J.N. Wilson W.R. Baddour L.M. Temporal trends in infective endocarditis: a population-based study in Olmsted County, Minnesota.JAMA. 2005; 293: 3022-3028Crossref PubMed Scopus (269) Google Scholar). These infective endocarditis-associated pathogens often contain a gene encoding a serine-rich repeat adhesin that can mediate the attachment of each respective pathogen to human platelet glycans (7.Bensing B.A. López J.A. Sullam P.M. The Streptococcus gordonii surface proteins GspB and Hsa mediate binding to sialylated carbohydrate epitopes on the platelet membrane glycoprotein Ibα.Infect. Immun. 2004; 72: 6528-6537Crossref PubMed Scopus (137) Google Scholar, 8.Plummer C. Wu H. Kerrigan S.W. Meade G. Cox D. Ian Douglas C.W. A serine-rich glycoprotein of Streptococcus sanguis mediates adhesion to platelets via GPIb.Br. J. Haematol. 2005; 129: 101-109Crossref PubMed Scopus (146) Google Scholar, 9.Takamatsu D. Bensing B.A. Cheng H. Jarvis G.A. Siboo I.R. Lopez J.A. Griffiss J.M. Sullam P.M. Binding of the Streptococcus gordonii surface glycoproteins GspB and Hsa to specific carbohydrate structures on platelet membrane glycoprotein Ibα.Mol. Microbiol. 2005; 58: 380-392Crossref PubMed Scopus (111) Google Scholar). The adhesive interaction is perhaps best understood in S. gordonii strain M99, which interacts with platelets via the serine-rich repeat adhesin GspB. This surface component mediates host adherence via a binding region that promotes a high affinity interaction to a narrow range of sialylated carbohydrates (10.Deng L. Bensing B.A. Thamadilok S. Yu H. Lau K. Chen X. Ruhl S. Sullam P.M. Varki A. Oral streptococci utilize a Siglec-like domain of serine-rich repeat adhesins to preferentially target platelet sialoglycans in human blood.PLoS Pathog. 2014; 10: e1004540Crossref PubMed Scopus (61) Google Scholar). Studies using recombinant GspB and whole bacteria have shown that the binding of GspB to the sialylated glycans of GPIbα is the primary adhesive interaction to host platelets (7.Bensing B.A. López J.A. Sullam P.M. The Streptococcus gordonii surface proteins GspB and Hsa mediate binding to sialylated carbohydrate epitopes on the platelet membrane glycoprotein Ibα.Infect. Immun. 2004; 72: 6528-6537Crossref PubMed Scopus (137) Google Scholar, 9.Takamatsu D. Bensing B.A. Cheng H. Jarvis G.A. Siboo I.R. Lopez J.A. Griffiss J.M. Sullam P.M. Binding of the Streptococcus gordonii surface glycoproteins GspB and Hsa to specific carbohydrate structures on platelet membrane glycoprotein Ibα.Mol. Microbiol. 2005; 58: 380-392Crossref PubMed Scopus (111) Google Scholar, 11.Takamatsu D. Bensing B.A. Prakobphol A. Fisher S.J. Sullam P.M. Binding of the streptococcal surface glycoproteins GspB and Hsa to human salivary proteins.Infect. Immun. 2006; 74: 1933-1940Crossref PubMed Scopus (81) Google Scholar) and that expression of GspB enhances virulence (12.Xiong Y.Q. Bensing B.A. Bayer A.S. Chambers H.F. Sullam P.M. Role of the serine-rich surface glycoprotein GspB of Streptococcus gordonii in the pathogenesis of infective endocarditis.Microb. Pathog. 2008; 45: 297-301Crossref PubMed Scopus (83) Google Scholar). S. sanguinis is often cited as the most common cause of bacterial infective endocarditis and contains a sequence homolog of GspB (8.Plummer C. Wu H. Kerrigan S.W. Meade G. Cox D. Ian Douglas C.W. A serine-rich glycoprotein of Streptococcus sanguis mediates adhesion to platelets via GPIb.Br. J. Haematol. 2005; 129: 101-109Crossref PubMed Scopus (146) Google Scholar). In S. sanguinis strain SK36, this homolog is called SrpA and includes a binding region (termed SrpABR) that is 32% identical and 46% similar to the corresponding region of GspB (termed GspBBR), but is significantly shorter, suggesting that GspBBR contains an additional domain as compared with SrpABR. Experimental validation of a role for SrpA in virulence is less clear than it is for GspB. Targeted mutagenesis of SrpA and other possible adhesins of S. sanguinis indicated that the deletion of SrpA does not influence virulence in an animal model of infective endocarditis (13.Turner L.S. Kanamoto T. Unoki T. Munro C.L. Wu H. Kitten T. Comprehensive evaluation of Streptococcus sanguinis cell wall-anchored proteins in early infective endocarditis.Infect. Immun. 2009; 77: 4966-4975Crossref PubMed Scopus (33) Google Scholar). However, the subsequent finding that individual deletion of every identified cell wall anchored protein in S. sanguinis had no significant influence on virulence suggests that S. sanguinis could have multiple adhesins that provide functional redundancy (13.Turner L.S. Kanamoto T. Unoki T. Munro C.L. Wu H. Kitten T. Comprehensive evaluation of Streptococcus sanguinis cell wall-anchored proteins in early infective endocarditis.Infect. Immun. 2009; 77: 4966-4975Crossref PubMed Scopus (33) Google Scholar). Like GspB, SrpA binds platelet glycoprotein GPIbα and can mediate binding to platelets in vitro (8.Plummer C. Wu H. Kerrigan S.W. Meade G. Cox D. Ian Douglas C.W. A serine-rich glycoprotein of Streptococcus sanguis mediates adhesion to platelets via GPIb.Br. J. Haematol. 2005; 129: 101-109Crossref PubMed Scopus (146) Google Scholar). Unlike GspB, SrpA appears to have a wider range of glycan-binding partners, but quantification of binding between SrpA and defined sialoglycans suggests only weak interactions for the carbohydrates tested (10.Deng L. Bensing B.A. Thamadilok S. Yu H. Lau K. Chen X. Ruhl S. Sullam P.M. Varki A. Oral streptococci utilize a Siglec-like domain of serine-rich repeat adhesins to preferentially target platelet sialoglycans in human blood.PLoS Pathog. 2014; 10: e1004540Crossref PubMed Scopus (61) Google Scholar). Sialoglycan array data further indicate that although GspB strongly prefers the N-acetylneuraminic acid (Neu5Ac) form of sialic acid, SrpA has some preference for glycans containing the N-glycolylneuraminic acid (Neu5Gc) form of sialic acid (10.Deng L. Bensing B.A. Thamadilok S. Yu H. Lau K. Chen X. Ruhl S. Sullam P.M. Varki A. Oral streptococci utilize a Siglec-like domain of serine-rich repeat adhesins to preferentially target platelet sialoglycans in human blood.PLoS Pathog. 2014; 10: e1004540Crossref PubMed Scopus (61) Google Scholar), which is not present in humans. Nevertheless, SrpA binds human platelets with high affinity (8.Plummer C. Wu H. Kerrigan S.W. Meade G. Cox D. Ian Douglas C.W. A serine-rich glycoprotein of Streptococcus sanguis mediates adhesion to platelets via GPIb.Br. J. Haematol. 2005; 129: 101-109Crossref PubMed Scopus (146) Google Scholar); thus it is possible that a high affinity human carbohydrate ligand for SrpA remains unidentified. The naturally occurring range of carbohydrate binding properties of GspB and SrpA can offer insights into the adhesive properties important for endovascular infection and may allow broader conclusions to be drawn about carbohydrate binding in these related adhesins. In previous work, we determined the crystal structure of the carbohydrate-binding region of GspB (14.Pyburn T.M. Bensing B.A. Xiong Y.Q. Melancon B.J. Tomasiak T.M. Ward N.J. Yankovskaya V. Oliver K.M. Cecchini G. Sulikowski G.A. Tyska M.J. Sullam P.M. Iverson T.M. A structural model for binding of the serine-rich repeat adhesin GspB to host carbohydrate receptors.PLoS Pathog. 2011; 7: e1002112Crossref PubMed Scopus (65) Google Scholar, 15.Pyburn T.M. Yankovskaya V. Bensing B.A. Cecchini G. Sullam P.M. Iverson T.M. Purification, crystallization and preliminary x-ray diffraction analysis of the carbohydrate-binding region of the Streptococcus gordonii adhesin GspB.Acta Crystallogr. Sect. F Struct. Biol. Cryst. Commun. 2010; 66: 1503-1507Crossref PubMed Scopus (4) Google Scholar). This binding region (termed GspBBR) contains three linearly arranged, independently folded domains termed the CnaA, Siglec, and Unique domains based upon their structural similarity to other proteins. The binding pocket for sialylated carbohydrates is located within the Siglec domain. Site-directed mutagenesis of residues in the GspB binding pocket identified residues critical for both carbohydrate binding and platelet binding, as well as for virulence in the setting of infective endocarditis (14.Pyburn T.M. Bensing B.A. Xiong Y.Q. Melancon B.J. Tomasiak T.M. Ward N.J. Yankovskaya V. Oliver K.M. Cecchini G. Sulikowski G.A. Tyska M.J. Sullam P.M. Iverson T.M. A structural model for binding of the serine-rich repeat adhesin GspB to host carbohydrate receptors.PLoS Pathog. 2011; 7: e1002112Crossref PubMed Scopus (65) Google Scholar). Here, we determine the crystal structure of the SrpA carbohydrate-binding region (SrpABR), both alone and in complex with a Neu5Gc-based synthetic sialyl galactoside disaccharide, and we use mutagenesis to validate key residues important for platelet binding. We identified a Thr-Arg motif conserved across homologous carbohydrate-binding adhesins that may orient the sialic acid moiety of carbohydrate ligands. Together, our results suggest that a small number of short sequence insertions in closely related homologs influence the quaternary structure, the tertiary structure, and the binding pocket. The latter may contribute to the differences in carbohydrate affinity and binding spectrum between SrpA and GspB. A codon-optimized gene for S. sanguinis strain SK36 SrpABR (encoding residues 240–453 of the full protein) was synthesized (DNA 2.0) and subcloned into the isopropyl 1-thio-β-d-galactopyranoside-inducible pSV278 vector (Vanderbilt University), which encodes a His6-maltose-binding protein (MBP) 3The abbreviations used are: MBP, maltose-binding protein; r.m.s., root mean square; DPBS, Dulbecco's PBS; GalβOMe, methyl β-galactopyranoside; ULI, unique loop insertion. affinity tag at the N terminus followed by a thrombin cleavage site. Variants of SrpABR were cloned into pGEX5X, which encodes an N-terminal GST tag followed by a factor Xa protease cleavage site. Wild type and variant SrpABR were expressed using the same protocol in Escherichia coli BL21 Gold (DE3) in LB medium and 50 μg/ml kanamycin or 100 μg ampicilllin, as appropriate for each plasmid. The cells were first grown at 37 °C until an A600 ∼0.5 was reached and then were cold shocked in an ice bath for 20 min. The cells were then moved to 18 °C, induced with 0.5 mm isopropyl 1-thio-β-d-galactopyranoside, and grown for 18 h. Cells were harvested by centrifugation for 15 min at 5000 × g at 4 °C and frozen at −20 °C before purification. Each frozen pellet was resuspended in binding buffer (20 mm Tris, pH 7.5, 200 mm NaCl, 1 mm EDTA) containing 1 mm PMSF, 2 μg/ml leupeptin, 2 μg/ml pepstatin, 1 μg/ml DNase, and 1 μg/ml RNase. The resuspended cells were then lysed by sonication. The lysate was clarified by centrifugation at 15,000 × g for 45 min and filtered using a 0.45-μm filter. Purification was performed at 4 °C. For wild-type SrpABR, the His6-MBP-SrpABR fusion protein was first purified by affinity chromatography with an MBP-Trap column. The eluted protein was concentrated in a 10-kDa cutoff concentrator and exchanged into 20 mm Tris, pH 7.5, and 200 mm NaCl. The His6-MBP affinity tag was then cleaved with 1 unit of thrombin per mg of protein and separated from SrpABR by passing the cleavage products over the MBP-Trap column in binding buffer. Variant SrpABR was first purified on a GST-Trap column and concentrated, and then the GST tag was cleaved with factor Xa protease. GST was selectively removed from the cleavage products by passage over the GST-Trap column. For both wild-type and variant protein, the protein aggregates and excess affinity tag were removed using a 24-ml Superdex S-200 size exclusion column in 20 mm Tris-HCl, pH 7.5, and 200 mm NaCl. For some protein preparations, separation of SrpABR from the fusion tag required an additional iteration of either affinity or size exclusion chromatography. After purification, the protein was >95% pure as assessed by visual inspection of the protein separated by SDS-PAGE. The SrpABR variant gene sequences, along with BamHI and EcoRI linkers, were synthesized (Life Technologies, Inc.) and then cloned in pGEX5X. The presence of only the expected alteration was verified by DNA sequence analysis. The GST-SrpABR fusion proteins were expressed and purified as described (10.Deng L. Bensing B.A. Thamadilok S. Yu H. Lau K. Chen X. Ruhl S. Sullam P.M. Varki A. Oral streptococci utilize a Siglec-like domain of serine-rich repeat adhesins to preferentially target platelet sialoglycans in human blood.PLoS Pathog. 2014; 10: e1004540Crossref PubMed Scopus (61) Google Scholar). To assess SrpABR binding to platelets, fresh human platelets were washed, fixed, and immobilized in 96-well plates as described (16.Bensing B.A. Sullam P.M. An accessory sec locus of Streptococcus gordonii is required for export of the surface protein GspB and for normal levels of binding to human platelets.Mol. Microbiol. 2002; 44: 1081-1094Crossref PubMed Scopus (203) Google Scholar). All subsequent binding steps were carried at out room temperature. To reduce nonspecific adherence, the wells were treated with 50 μl of a 1× casein solution (blocking reagent, Roche Applied Science) in Dulbecco's PBS (DPBS) for 1 h. The blocking solution was replaced with 50 μl of purified GST-SrpABR wild-type or variant proteins (0.5 μm in 1× blocking solution). The plates were incubated for 1 h with vigorous rocking; wells were rinsed three times with 100 μl of DPBS, and 50 μl of a rabbit polyclonal anti-GST (Life Technologies, Inc.) diluted 1:500 in 1× blocking solution was added to each well. After 1 h, wells were rinsed three times with 100 μl of DPBS, and 50 μl of a peroxidase-conjugated anti-rabbit antibody (1:5000 dilution in DPBS) was added. After incubation for 1 h, wells were rinsed three times with 100 μl of DPBS, and 200 μl of a solution of 0.4 mg/ml o-phenylenediamine dihydrochloride (Sigma) was added. The absorbance at 450 nm was read after ∼15 min. Results are reported as the mean ± S.D., with n = 4 for each protein tested. GalβOMe (50 mg, 0.26 mmol), N-glycolylmannosamine (91 mg, 0.38 mmol), sodium pyruvate (113 mg, 1.03 mmol), and CTP (217 mg, 0.38 mmol) were dissolved in Tris-HCl buffer (10 ml, 100 mm, pH 8.5) containing MgCl2 (20 mm) and appropriate amounts of Pasteurella multocida sialic acid aldolase (3 mg), Neisseria meningitidis CMP-sialic acid synthetase (2 mg), and Pasteurella multocida sialyltransferase 1 M144D (2 mg). The reaction was carried out by incubating the reaction mixture in an incubator shaker at 37 °C for 12 h. The reaction was monitored by TLC (EtOAc/MeOH/H2O/HOAc = 5:2:1:0.1, by volume) with p-anisaldehyde sugar staining and mass spectrometry. When an optimal yield was achieved, to the reaction mixture was added the same volume (10 ml) of 95% ethanol followed by incubation at 4 °C for 30 min. The precipitates were removed by centrifugation (5533 × g, 30 min), and the supernatant was concentrated and purified by Bio-Gel P-2 gel filtration chromatography (water was used as an eluent). Further purification was achieved using silica gel chromatography (EtOAc/MeOH/H2O = 4:2:1, by volume) and a final pass through of a Bio-Gel P-2 gel filtration column to produce Neu5Gcα2–3GalβOMe (101 mg). Yield, 81%; white foam. 1H NMR (800 MHz, D2O) δ 4.38 (d, J = 8.0 Hz, 1H), 4.10 (s, 2H), 4.08 (dd, J = 9.6 and 3.2 Hz, 1H), 3.93 (d, J = 2.8 Hz, 1H), 3.91 (t, J = 10.4 Hz, 1H), 3.86–3.58 (m, 10H), 3.56 (s, 3H), 3.53 (t, J = 8.8 Hz, 1H), 2.75 (dd, J = 12.8 and 4.8 Hz, 1H), 1.79 (t, 1H, J = 12.0 Hz, 1H); 13C NMR (200 MHz, D2O) δ 175.66, 173.76, 103.34, 99.70, 75.72, 74.81, 73.43, 72.44, 71.73, 69.02, 67.96, 67.85, 67.40, 62.38, 60.84, 56.91, 51.25, 39.60; high resolution mass spectrometry (ESI) m/z calculated for C18H30O15N (M − H) 500.1615, found 500.1608. SrpABR was concentrated and buffer exchanged into 20 mm Tris-HCl, pH 7.2. All crystals grewusing the sitting drop vapor diffusion method in a CombiClover 4 chamber plate at room temperature (∼23 °C) by equilibrating droplets containing 1 μl of protein and 1 μl of reservoir solution over 50 μl of a reservoir solution. Native crystals grew in space group C2 using a protein concentration of 10.6 mg/ml and a reservoir condition that contained 0.2 m Ca(CH3CO2)2, 0.1 m sodium cacodylate, pH 6.5, and 18% PEG 8000. Cocrystals of SrpABR with the Neu5Gcα2–3GalβOMe synthetic disaccharide used 14.4 mg/ml protein in a buffer solution containing 10 mm carbohydrate and 20 mm Tris-HCl, pH 7.2. Crystals of the SrpABR R347E variant were grown using a protein concentration of 15.3 mg/ml and a reservoir solution containing 0.2 m NaCH3COO, 0.1 m sodium cacodylate, pH 6.5, and 30% PEG 8000. An Os derivative was generated by soaking fully formed crystals in the C2 space group in reservoir solution supplemented with 2 mm K2OsO4 and 5% DMSO overnight. Native crystals were cryo-protected using a solution containing all of the components of the reservoir solution plus 5% DMSO before cryo-cooling while the Os derivative crystals were flash-cooled directly from the soaking drop, and disaccharide cocrystals were cryo-cooled using the well solution supplemented with 5 mm disaccharide. Diffraction data were collected using the LS-CAT beamlines of the Advanced Photon Source at −180 °C as listed in Table 1. Data were processed using the HKL2000 (17.Otwinowski Z. Minor W. Processing of x-ray diffraction data collected in oscillation mode.Methods Enzymol. 1997; 276: 307-326Crossref PubMed Scopus (38527) Google Scholar) and CCP4 (18.Winn M.D. Ballard C.C. Cowtan K.D. Dodson E.J. Emsley P. Evans P.R. Keegan R.M. Krissinel E.B. Leslie A.G. McCoy A. McNicholas S.J. Murshudov G.N. Pannu N.S. Potterton E.A. Powell H.R. et al.Overview of the CCP4 suite and current developments.Acta Crystallogr. D Biol. Crystallogr. 2011; 67: 235-242Crossref PubMed Scopus (9206) Google Scholar) suites of programs.TABLE 1Crystallographic data collection and refinement statisticsSrpABRSrpABR (Os)SrpABR Neu5Gcα2–3GalβOMeSrpABR R347EPDB entry5EQ2NA5EQ35EQ4Data collectionBeamlineID-FID-DID-GID-GWavelength0.979 Å1.13966 Å0.979 Å0.979 ÅSpace groupC2C2C2P212121Unit cella = 174.5 Åa = 174.4 Åa = 174.6 Åa = 44.7 Åb = 46.8 Åb = 46.8 Åb = 47.0 Åb = 88.1 Åc = 64.8 Åc = 64.7 Åc = 64.0 Åc = 199.2 Åβ = 102.7°β = 102.8°β = 102.2°Resolution1.8 Å2.2 Å2.0 Å2.3 ÅRsym0.061 (0.450)0.101 (0.467)0.131 (0.500)0.095 (0.563)Rpim0.034 (0.244)0.029 (0.188)0.054 (0.224)0.045 (0.316)I/σ30.9 (4.8)34.1 (5.1)23.0 (2.9)14.2 (2.2)Completeness (%)98.8 (100)100 (100)94.7 (78.0)91.8 (71.6)Redundancy4.0 (4.2)12.5 (7.0)6.8 (5.3)4.8 (3.6)CC1/20.9600.9350.8810.759RefinementNo. of molecules/asymmetric unit224Rcryst0.1780.1980.202Rfree0.2080.2430.257r.m.s. deviationBond lengths0.01 Å0.007 Å0.002 ÅBond angles1.15°0.98°0.59°RamachandranFavored97.7%96.8%96.6%Allowed2.3%3.2%3.1%Outliers0%0%0.3%Average B-factor31.54 Å235.21 Å230.94 Å2Ligand B-factor—42.58 Å2 Open table in a new tab The structure in the C2 space group was determined using single wavelength anomalous diffraction phasing from the Os derivative. Eleven Os sites were identified using the AutoSol subroutine in Phenix (19.Adams P.D. Afonine P.V. Bunkóczi G. Chen V.B. Davis I.W. Echols N. Headd J.J. Hung L.W. Kapral G.J. Grosse-Kunstleve R.W. McCoy A.J. Moriarty N.W. Oeffner R. Read R.J. Richardson D.C. et al.PHENIX: a comprehensive Python-based system for macromolecular structure solution.Acta Crystallogr. D Biol. Crystallogr. 2010; 66: 213-221Crossref PubMed Scopus (16439) Google Scholar, 20.Terwilliger T.C. Adams P.D. Read R.J. McCoy A.J. Moriarty N.W. Grosse-Kunstleve R.W. Afonine P.V. Zwart P.H. Hung L.W. Decision-making in structure solution using Bayesian estimates of map quality: the PHENIX AutoSol wizard.Acta Crystallogr. D Biol. Crystallogr. 2009; 65: 582-601Crossref PubMed Scopus (673) Google Scholar), and phases were calculated using a f″ of 10.2 and an f′ of −17.65 with an overall figure of merit of 0.221. Phases were improved using the Resolve subroutine in Phenix (19.Adams P.D. Afonine P.V. Bunkóczi G. Chen V.B. Davis I.W. Echols N. Headd J.J. Hung L.W. Kapral G.J. Grosse-Kunstleve R.W. McCoy A.J. Moriarty N.W. Oeffner R. Read R.J. Richardson D.C. et al.PHENIX: a comprehensive Python-based system for macromolecular structure solution.Acta Crystallogr. D Biol. Crystallogr. 2010; 66: 213-221Crossref PubMed Scopus (16439) Google Scholar), and the initial model was built using the Buccaneer subroutine in Phenix (19.Adams P.D. Afonine P.V. Bunkóczi G. Chen V.B. Davis I.W. Echols N. Headd J.J. Hung L.W. Kapral G.J. Grosse-Kunstleve R.W. McCoy A.J. Moriarty N.W. Oeffner R. Read R.J. Richardson D.C. et al.PHENIX: a comprehensive Python-based system for macromolecular structure solution.Acta Crystallogr. D Biol. Crystallogr. 2010; 66: 213-221Crossref PubMed Scopus (16439) Google Scholar, 21.Cowtan K. The Buccaneer software for automated model building. 1. Tracing protein chains.Acta Crystallogr. D Biol. Crystallogr. 2006; 62: 1002-1011Crossref PubMed Scopus (1459) Google Scholar). This procedure identified two molecules in the asymmetric unit and resulted in a model with an Rfree of 0.24. The initial coordinates then transferred directly into the native data set with an initial round of rigid body refinement. The unliganded and disaccharide-bound crystals of SrpABR were isomorphous, and thus the disaccharide-bound structure was determined by isomorphous replacement. In short, following refinement of the unliganded structure, all solvent molecules were removed, and the coordinates were transferred directly into the dataset for SrpABR cocrystallized with the disaccharide. The coordinates were subjected to rigid body refinement in Phenix (19.Adams P.D. Afonine P.V. Bunkóczi G. Chen V.B. Davis I.W. Echols N. Headd J.J. Hung L.W. Kapral G.J. Grosse-Kunstleve R.W. McCoy A.J. Moriarty N.W. Oeffner R. Read R.J. Richardson D.C. et al.PHENIX: a comprehensive Python-based system for macromolecular structure solution.Acta Crystallogr. D Biol. Crystallogr. 2010; 66: 213-221Crossref PubMed Scopus (16439) Google Scholar), resulting in an initial Rfree of 0.27. Unambiguous electron density for the sialic acid was observed even in the initial electron density maps, and both the sialic acid and the galactose were placed into the model after two rounds of refinement. The occupancy of the disaccharide was retained at 1.0 for the duration of the refinement, and the average temperature factors of this ligand (42.6 Å2) are similar to that of the protein (35.2 Å2) in the final model. The structure of the R347E variant in the P212121 space group was determined by molecular replacement using the Phaser subroutine in Phenix and the coordinates of the unliganded protein with all solvent molecules and ligands removed. The final model of the R347E variant contained a geometric outlier (Table 1) within the loop that changed position upon mutation. This region contains electron density that was challenging to assign with confidence. All models were improved with iterative rounds of model building in Coot (22.Emsley P. Cowtan K. Coot: model-building tools for molecular graphics.Acta Crystallogr. D Biol. Crystallogr. 2004; 60: 2126-2132Crossref PubMed Scopus (23229) Google Scholar) and refinement in Phenix (19.Adams P.D. Afonine P.V. Bunkóczi G. Chen V.B. Davis I.W. Echols N. Headd J.J. Hung L.W. Kapral G.J. Grosse-Kunstleve R.W. McCoy A.J. Moriarty N.W. Oeffner R. Read R.J. Richardson D.C. et al.PHENIX: a comprehensive Python-based system for macromolecular structure solution.Acta Crystallogr. D Biol. Crystallogr. 2010; 66: 213-221Crossref PubMed Scopus (16439) Google Scholar). Domain rotation analysis was performed using the DynDoM web server (23.Poornam G.P. Matsumoto A. Ishida H. Hayward S. A method for the analysis of domain movements in large biomolecular complexes.Proteins. 2009; 76: 201-212Crossref PubMed Scopus (80) Google Scholar). Dynamic light scattering was performed using a DynaPro NanoStar instrument (Wyatt Technology) on protein samples that were first filtered through a 0.22-μm Spin-X centrifugal filter (Corning Inc.). Measurements were performed in triplicate with an SrpABR concentration of 1 mg/ml in 20 mm sodium cacodylate, 10 mm CaCl2, pH 7.2, at 25 °C. Three measurements of 10 acquisitions were collected for each of three SrpABR samples. Dynamics 7.1.9 software was used for data analysis. The x-ray crystal structure of unliganded SrpABR (residues 240–453 of the full-length protein) was determined to 1.8 Å resolution (Table 1) using de novo phasing calculated from single wavelength anomalous diffraction of an osmium derivative. Each SrpABR protomer folds into two domains (Fig. 1). The N-terminal domain is similar to the “Siglec”-like domain of GspB that is a variant of a V-set Ig fold, with an r.m.s. deviation of 1.8 Å over the 102 amino acids in the alignment. The C-terminal domain is similar to the “Unique” domain of GspB with an r.m.s. deviation of 1.4 Å over the 76 amino acids in the alignment. Given the close conservation of fold in each domain, we used these structures to improve the accuracy of our sequence alignment with GspB (Fig. 2). This sequence alignment reveals several notable insertions in each adhesin with respect to each other. These insertions add structural elements to the surface of SrpA (Fig. 2B) or GspB (Fig. 2C) that appear to alter the structural and functional properties of the adhesins.FIGURE 2Structure-based sequence alignment of SrpA and GspB. A, sequence alignment was performed manually by overlaying the structures of SrpABR and GspBBR. 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" @default.
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• W2272772088 date "2016-04-01" @default.
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• W2272772088 title "Structural Basis for Sialoglycan Binding by the Streptococcus sanguinis SrpA Adhesin" @default.
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