Sialic Acid Identity Modulates Host Tropism of Sialoglycan-binding Viridans Group Streptococci

Sialic Acid Identity Modulates Host Tropism of Sialoglycan-binding Viridans Group Streptococci | bioRxiv /* */ /* */ <!-- <!-- /*! * yepnope1.5.4 * (c) WTFPL, GPLv2 */ (function(a,b,c){function d(a){return"[object Function]"==o.call(a)}function e(a){return"string"==typeof a}function f(){}function g(a){return!a||"loaded"==a||"complete"==a||"uninitialized"==a}function h(){var a=p.shift();q=1,a?a.t?m(function(){("c"==a.t?B.injectCss:B.injectJs)(a.s,0,a.a,a.x,a.e,1)},0):(a(),h()):q=0}function i(a,c,d,e,f,i,j){function k(b){if(!o&&g(l.readyState)&&(u.r=o=1,!q&&h(),l.onload=l.onreadystatechange=null,b)){"img"!=a&&m(function(){t.removeChild(l)},50);for(var d in y[c])y[c].hasOwnProperty(d)&&y[c][d].onload()}}var j=j||B.errorTimeout,l=b.createElement(a),o=0,r=0,u={t:d,s:c,e:f,a:i,x:j};1===y[c]&&(r=1,y[c]=[]),"object"==a?l.data=c:(l.src=c,l.type=a),l.width=l.height="0",l.onerror=l.onload=l.onreadystatechange=function(){k.call(this,r)},p.splice(e,0,u),"img"!=a&&(r||2===y[c]?(t.insertBefore(l,s?null:n),m(k,j)):y[c].push(l))}function j(a,b,c,d,f){return q=0,b=b||"j",e(a)?i("c"==b?v:u,a,b,this.i++,c,d,f):(p.splice(this.i++,0,a),1==p.length&&h()),this}function k(){var a=B;return a.loader={load:j,i:0},a}var l=b.documentElement,m=a.setTimeout,n=b.getElementsByTagName("script")[0],o={}.toString,p=[],q=0,r="MozAppearance"in l.style,s=r&&!!b.createRange().compareNode,t=s?l:n.parentNode,l=a.opera&&"[object Opera]"==o.call(a.opera),l=!!b.attachEvent&&!l,u=r?"object":l?"script":"img",v=l?"script":u,w=Array.isArray||function(a){return"[object Array]"==o.call(a)},x=[],y={},z={timeout:function(a,b){return b.length&&(a.timeout=b[0]),a}},A,B;B=function(a){function b(a){var a=a.split("!"),b=x.length,c=a.pop(),d=a.length,c={url:c,origUrl:c,prefixes:a},e,f,g;for(f=0;f<d;f++)g=a[f].split("="),(e=z[g.shift()])&&(c=e(c,g));for(f=0;f<b;f++)c=x[f](c);return c}function g(a,e,f,g,h){var i=b(a),j=i.autoCallback;i.url.split(".").pop().split("?").shift(),i.bypass||(e&&(e=d(e)?e:e[a]||e[g]||e[a.split("/").pop().split("?")[0]]),i.instead?i.instead(a,e,f,g,h):(y[i.url]?i.noexec=!0:y[i.url]=1,f.load(i.url,i.forceCSS||!i.forceJS&&"css"==i.url.split(".").pop().split("?").shift()?"c":c,i.noexec,i.attrs,i.timeout),(d(e)||d(j))&&f.load(function(){k(),e&&e(i.origUrl,h,g),j&&j(i.origUrl,h,g),y[i.url]=2})))}function h(a,b){function c(a,c){if(a){if(e(a))c||(j=function(){var a=[].slice.call(arguments);k.apply(this,a),l()}),g(a,j,b,0,h);else if(Object(a)===a)for(n in m=function(){var b=0,c;for(c in a)a.hasOwnProperty(c)&&b++;return b}(),a)a.hasOwnProperty(n)&&(!c&&!--m&&(d(j)?j=function(){var a=[].slice.call(arguments);k.apply(this,a),l()}:j[n]=function(a){return function(){var b=[].slice.call(arguments);a&&a.apply(this,b),l()}}(k[n])),g(a[n],j,b,n,h))}else!c&&l()}var h=!!a.test,i=a.load||a.both,j=a.callback||f,k=j,l=a.complete||f,m,n;c(h?a.yep:a.nope,!!i),i&&c(i)}var i,j,l=this.yepnope.loader;if(e(a))g(a,0,l,0);else if(w(a))for(i=0;i (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0];var j=d.createElement(s);var dl=l!='dataLayer'?'&l='+l:'';j.src='//www.googletagmanager.com/gtm.js?id='+i+dl;j.type='text/javascript';j.async=true;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-M677548'); Skip to main content Home About Submit ALERTS / RSS Search for this keyword Advanced Search New Results Sialic Acid Identity Modulates Host Tropism of Sialoglycan-binding Viridans Group Streptococci KeAndreya M. Morrison , Rupesh Agarwal , Haley E. Stubbs , Hai Yu , Stefan Ruhl , Xi Chen , Paul M Sullam , Barbara A Bensing , Jeremy C. Smith , View ORCID Profile T. M. Iverson doi: https://doi.org/10.1101/2025.06.24.660003 KeAndreya M. Morrison 1 Department of Pharmacology, School of Graduate Studies, Meharry Medical College , Nashville, TN, USA Find this author on Google Scholar Find this author on PubMed Search for this author on this site Rupesh Agarwal 2 UT/ORNL Center for Molecular Biophysics, Oak Ridge National Laboratory , TN, USA Find this author on Google Scholar Find this author on PubMed Search for this author on this site Haley E. Stubbs 3 Chemical and Physical Biology Program, Vanderbilt University , Nashville, TN, USA Find this author on Google Scholar Find this author on PubMed Search for this author on this site Hai Yu 4 Department of Chemistry, University of California , Davis, CA, USA Find this author on Google Scholar Find this author on PubMed Search for this author on this site Stefan Ruhl 5 Department of Oral Biology, University at Buffalo , Buffalo, NY, USA Find this author on Google Scholar Find this author on PubMed Search for this author on this site Xi Chen 4 Department of Chemistry, University of California , Davis, CA, USA Find this author on Google Scholar Find this author on PubMed Search for this author on this site Paul M Sullam 6 Division of Infectious Diseases, Veterans Affairs Medical Center, Department of Medicine, University of California , San Francisco, and the Northern California Institute for Research and Education , San Francisco, CA, USA Find this author on Google Scholar Find this author on PubMed Search for this author on this site Barbara A Bensing 6 Division of Infectious Diseases, Veterans Affairs Medical Center, Department of Medicine, University of California , San Francisco, and the Northern California Institute for Research and Education , San Francisco, CA, USA Find this author on Google Scholar Find this author on PubMed Search for this author on this site Jeremy C. Smith 2 UT/ORNL Center for Molecular Biophysics, Oak Ridge National Laboratory , TN, USA 7 Department of Biochemistry and Cellular and Molecular Biology, University of Tennessee , Knoxville, TN Find this author on Google Scholar Find this author on PubMed Search for this author on this site T. M. Iverson 1 Department of Pharmacology, School of Graduate Studies, Meharry Medical College , Nashville, TN, USA 8 Departments of Pharmacology and Biochemistry, Vanderbilt University , Nashville, TN, USA Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for T. M. Iverson For correspondence: tina.iverson{at}vanderbilt.edu Abstract Full Text Info/History Metrics Supplementary material Preview PDF Abstract Microbial interactions with multiple species may expand the range of potential hosts, supporting both pathogen reservoirs and zoonotic spillover. Viridans group streptococci interact with host cells by engaging protein-attached glycosylations capped with terminal sialic acids (sialoglycans). One potential origin for host tropism of these streptococci arises because humans exclusively synthesize the N-acetylneuraminic acid (Neu5Ac) form of sialic acid, while non-human mammals synthesize both Neu5Ac and a hydroxylated N-glycolylneuraminic acid (Neu5Gc). However, the link between binding preference for these sialic acids and preference for host has not been tested experimentally. Here, we investigate sialoglycan-binding by Neu5Ac/Neu5Gc cross-reactive Siglec-like binding regions (SLBRs) from two strains of streptococci, Streptococcus gordonii strain Challis (SLBR Hsa ) and Streptococcus sanguinis strain SK36 (SLBR SrpA ). Structural and computational analyses of SLBR Hsa identified molecular details for the binding of disaccharides capped in Neu5Ac or Neu5Gc. Engineering SLBR Hsa and SLBR SrpA for narrow selectivity to synthetic Neu5Gc-terminated glycans shifted the binding preference from authentic human plasma receptors to plasma receptors from rat sources. However, host receptor preference did not fully recapitulate purified Neu5Ac/Neu5Gc-capped sialoglycan preference. These findings suggest that sialic acid identity modulates, but does not uniquely determine, host preference by these streptococci. This work refines our understanding of host specificity and challenges prevailing assumptions about the relative role of sialic acids in host tropism. Introduction Viridans group streptococci are among the bacteria that can engage sialic acid capped glycans (sialoglycans) on host cells 1 . This host adherence promotes oral colonization when the sialic acid is attached to the glycans on salivary proteins, such as MUC7 2 – 4 . Host adherence also supports endovascular pathogenesis 5 , 6 . Indeed, engagement of sialoglycans attached to platelet glycoprotein Ibα 7 (GPIbα) is among the first committed steps in bacterial infective endocarditis, a serious infection of the heart valves that may result in heart failure, stroke, or permanent valve damage, even with aggressive treatment. Viridans group streptococci are responsible for 40%-60% of bacterial infective endocarditis cases 8 , 9 . In-hospital mortality for these infections is approximately 10%, while one- and five-year mortality rates are estimated as 22-37% and 37-53%, respectively 10 – 13 . Streptococci bind to sialoglycans using an adhesin that contains a domain related to mammalian S ialic acid-binding immuno g lobulin(Ig)-like lec tins (Siglecs) 14 . In streptococci, this binding domain is called the Siglec-like binding region (SLBR) 14 . Both SLBRs and Siglecs are organized around a V-set Ig fold that is predominantly comprised of β-strands and that has a standard nomenclature. The strands of the Ig fold are designated A-G and intervening loops are named based upon the adjacent strands 14 ; for example, the AB loop connects the A strand and the B strand. The sialoglycan itself binds above a conserved ΦTRX sequence motif on the F strand, where Φ is a hydrophobic amino acid, most commonly tyrosine. SLBRs engage sialic acid capped terminal tri- and tetra-saccharides 15 , 16 on heterogeneous glycosylations, which can subtly differ in identity and presentation between individuals 17 , 18 . Importantly, the preferred sialoglycan ligand of SLBRs correlates with disease severity in an animal model, with selective binding to a sialyl-T antigen-capped receptor on GPIbα most strongly promoting endocardial infection 6 . As revealed by chimeragenesis, control over the preferred sialoglycan ligand disproportionately involves direct interactions with side chains on three loops that surround the binding site: that CD loop, the EF loop, and the FG loop 16 . Perhaps surprisingly, it is difficult to predict the sialoglycan binding spectrum of SLBRs. Even SLBRs with >90% identity can exhibit a different sialoglycan binding repertoire 16 , 19 . Humans and non-human animals can differ in their production and presentation of sialoglycans. Within the context of complex glycosylations, these differences include the underlying glycan composition, the preferred glycosyl linkages, the local distribution of different sialoglycans in biological niches, and even the sialic acid itself 20 , 21 . Sialic acids are nine-carbon acidic sugars with more than 50 biological forms 17 , 21 , 22 . Whereas humans only synthesize N-acetylneuraminic acid (Neu5Ac, Fig. 1A ) and its derivatives, most non-human animals can convert Neu5Ac to N-glycolylneuraminic acid (Neu5Gc, Fig. 1B ) 23 and its derivatives. Neu5Ac and Neu5Gc only differ by a hydroxyl group appended at the C11 position 24 , 25 ( Fig. 1B ). This is subtle, yet some infectious agents can distinguish between the two 26 – 29 . Download figure Open in new tab Figure 1 Chemical structures of Neu5Ac- and Neu5Gc-terminated α2-3-linked sialic acid-galactose disaccharides A) α2-3-linked N -acetylneuraminic acid (Neu5Ac)-Galactose (Gal) (Neu5Acα2-3Gal). The C11 is highlighted. B) α2-3-linked N -glycolylneuraminic acid (Neu5Gc)-GalβOMe (Neu5Gcα2-3GalβOMe). The additional OH11 is highlighted. Here, we evaluate SLBRs from Streptococcus gordonii strain Challis (SLBR Hsa ) and Streptococcus sanguinis strain SK36 (SLBR SrpA ), both of which can strongly engage sialoglycans capped by either Neu5Ac or Neu5Gc. Using structural and computational approaches, we asked how these SLBRs can bind synthetic sialoglycans terminating in either Neu5Ac or Neu5Gc, how this affects sialoglycan binding preference in solution, and how this correlates with native sialoglycoside engagement. Our results reveal that Neu5Ac versus Neu5Gc preference modulates, but does not uniquely determine, host specificity. These findings refine our understanding of how these bacteria target their hosts, clarify molecular aspects of tropism, and may even provide initial insight into molecular drivers of bacterial species jumps. Results X-ray Crystal Structures of SLBR Hsa Bound to Neu5Ac- and Neu5Gc-Terminated Disaccharides To investigate how SLBR Hsa engages Neu5Ac versus Neu5Gc, we used X-ray crystallography. We determined X-ray crystal structures of SLBR Hsa bound to synthetic α2-3-linked disaccharides Neu5Gcα2-3GalβOMe (SLBR Hsa –Neu5Gc, Fig. 2A, 2B ) and Neu5Acα2-3Gal (SLBR Hsa –Neu5Ac, Fig. 2A, 2C ). The SLBR Hsa –Neu5Gc complex was determined at 1.30 Å resolution ( Fig. 2B , Table 1 ), and the SLBR Hsa –Neu5Ac complex was determined at 1.45 Å resolution ( Fig. 2C , Table 1 ). Note that the methyl aglycon in Neu5Gcα2-3GalβOMe ( Fig. 2B ) arises from its chemical synthesis 30 , 31 . This leaving group is found on a region of the glycan that does not directly contact the SLBR ( Fig. 2B ). Moreover, we do not observe any structural perturbations attributable to this feature here or in reported costructures of Neu5Gc with SLBR SrpA 32 , where X-ray crystal structures with both Neu5Gc- and Neu5Ac-terminated α2-3-linked disaccharides have been reported 32 , 33 . Download figure Open in new tab Figure 2 X-ray crystal structures of Neu5Acα2-3Gal or Neu5Gcα2-3GalβOMe bound to SLBR Hsa A) Ribbons diagram of SLBR Hsa with the strands of the V-set Ig fold labeled and the CD-( orange ), EF-( blue ), and FG-loops ( hot pink ) highlighted. Sialyl disaccharides are colored according to SNFG convention, with Neu5Ac in purple , Neu5Gc in cyan , and Gal in yellow . B) and C) Zoomed-in views rotated 45° around the x-axis to highlight hydrogen bonds between B) Neu5Acα2-3Gal or C) Neu5Gcα2-3GalβOMe and the ФTRX motif (SLBR Hsa Y338 , SLBR Hsa T339 , SLBR Hsa R340 , SLBR Hsa Y341 ) on the F-strand. Each model is superimposed with composite omit electron density ( green mesh ). View this table: View inline View popup Download powerpoint Table 1 X-ray crystallographic data collection and refinement statistics for SLBR Hsa bound to Neu5Acα2-3Gal or Neu5Gcα2-3GalβOMe Values in parentheses are for the highest resolution shell. Raw data are deposited with SBGrid and can be accessed at: data.sbgrid.org/dataset/DATAID . The positions and binding of these two sialyl disaccharide ligands are conserved between SLBR Hsa –Neu5Ac and SLBR Hsa –Neu5Gc, with superposition of the SLBR Hsa –Neu5Ac and SLBR Hsa –Neu5Gc showing that the binding position for each sialoglycan is within the error of the resolution. Each sialoglycan binds above the conserved ΦTRX motif of the F strand of the Siglec domain, where residues SLBR Hsa Y338 , SLBR Hsa T339 , SLBR Hsa R340 , and SLBR Hsa Y341 stabilize ligands with hydrogen-bonding interactions ( Fig. 2B, 2C ). This binding site is located between the CD, EF, and FG loops, making the overall binding mode similar to the Neu5Acα2-3Gal terminus of the sialyl tri- and tetra-saccharides in reported SLBR–sialoglycan complexes 2 , 16 ( Fig. 2B, 2C ). Structural comparison of SLBR Hsa –Neu5Ac and SLBR Hsa –Neu5Gc with the previously reported unliganded SLBR Hsa 16 also reveals similar overall folds, with RMSD values for Cα atoms of 0.157 Å (Neu5Ac-bound) and 0.158 Å (Neu5Gc-bound). One small difference is the position of the EF loop. Past work shows that this EF loop can close over bound tri- and tetrasaccharides 16 . In these structures, the EF loop closes over Neu5Gc but not over Neu5Ac, with a maximal backbone displacement of 4.6 Å ( Fig. 2B, 2C , Supplementary Fig. 1A ). Loop closure is unlikely to impact Neu5Ac versus Neu5Gc selectivity as it does not detectably affect contacts to C11 or the C11-appended hydroxyl (OH11). Moreover, past work using the same crystal form of SLBR Hsa identified that the EF loop is somewhat stabilized in the open position by crystal contacts 16 . The difference in loop position that is observed here more likely results from this same phenomenon rather than by differences in ligand. To provide more insight, we compared these SLBR Hsa structures to reported structures of SLBR SrpA , a related SLBR that can similarly bind sialoglycans terminating in either Neu5Ac or Neu5Gc 7 , 33 and where experimental structures with each ligand have been reported 32 ( Fig. 3 , Supplementary Fig. 1B, Supplementary Fig. 2 ). Comparison of SLBR Hsa –Neu5Gc with SLBR SrpA –Neu5Gc 32 shows that Neu5Gc similarly binds above the F strand in both these SLBRs, albeit with a lateral shift in position of 1.5 Å with respect to the ΦTRX motif ( Supplementary Fig. 1B ). An additional difference is a 160° rotation of OH11 between the two ligands ( Fig. 3A, 3B ). In SLBR Hsa –Neu5Gc, the OH11 orients toward solvent and does not interact with the protein ( Fig. 3A ). The closest protein atoms to OH11 are the SLBR Hsa Y338 side chain hydroxyl on the F-strand and SLBR Hsa S336 Oψ on the EF loop, with distances of 3.8 Å in each case ( Fig. 3A ). The closest atoms to C11 are the SLBR Hsa Y338 side chain hydroxyl and SLBR Hsa S336 Cβ, with distances of 3.2 Å and 3.9 Å, respectively ( Fig. 3A ). In comparison, a unique hydrogen bond forms between OH11 and the SLBR SrpA Y368 side chain hydroxyl that orients OH11 toward the protein ( Fig. 3B ). Comparison of SLBR Hsa –Neu5Ac ( Supplementary Fig. 2A ) with SLBR SrpA –Neu5Ac 32 ( Supplementary Fig. 2B ) shows the same 1.5 Å lateral shift in glycan position with respect to the ΦTRX motif as is observed for the Neu5Gc costructures. Download figure Open in new tab Figure 3 Structures of SLBR Hsa –Neu5Gc and SLBR SrpA –Neu5Gc Side-by-side comparison of Neu5Gc-GalβOMe binding in A) SLBR Hsa and B) SLBR SrpA 32 illustrates differences in the positioning of the C11 hydroxyl group (OH11) of Neu5Gc. A ) In SLBR Hsa , the residues adjacent to sialic acid include SLBR Hsa S336 and SLBR Hsa Y338 . These residues lack direct contact with the OH11 group, which is oriented away from the binding site. B ) In contrast, SLBR SrpA Y368 hydrogen-bonds to the OH11 group. Distances for adjacent non-bonding atoms are indicated with brackets.; the hydrogen-bond between SLBR SrpA Y368 OH and Neu5Gc OH11 is shown with a dotted line and marked with blue text. We next assessed whether there were differences between the Neu5Ac/Neu5Gc binding SLBRs and Neu5Ac-selective SLBRs ( Fig. 4 , Supplementary Fig. 2 ). For the Neu5Ac/Neu5Gc binding SLBRS, we used SLBR Hsa –Neu5Ac/Neu5Gc ( Fig. 4A , Supplementary Fig. 2A ), SLBR SrpA –Neu5Ac/Neu5Gc ( Fig. 4B , Supplementary Fig. 2B ) and an additional Neu5Ac/Neu5Gc binding comparator, SLBR UB10712 ( Fig. 4C ) where there is only a structure of the SLBR without ligand bound 16 . For the Neu5Ac-selective SLBRs, we used SLBR GspB –Neu5Ac 14 ( Fig. 4D , Supplementary Fig. 2C ), SLBR SK1 –Neu5Ac 34 ( Fig. 4E , Supplementary Fig. 2D ), and SLBR SK678 16 ( Fig. 4F ), which only has a structure of the SLBR without ligand bound 16 . This comparison identified that the Neu5Ac/Neu5Gc-binding SLBRs had a more open binding pocket near C11 and OH11 ( Fig. 4A , 4C , 4E ) while the Neu5Ac-selective SLBRS shows a more defined pocket that likely has more precise Van der Waals contacts near C11 ( Fig. 4B , 4D , 4F ). Other potential features such as electrostatics ( Fig. 4A - 4F ), bonding patterns, and water molecule substructure did not correlate with sialic acid selectivity. Download figure Open in new tab Figure 4 Surface rendering of SLBRs colored by electrostatic potential In the Figure, surfaces with positive charge are colored blue , surfaces with negative charge are colored red , and neutral surfaces are colored white . A) SLBR Hsa , B) SLBR SrpA 32 , C) SLBR UB19712 16 , D) SLBR GspB 14 , E) SLBR SK1 34 , and F) SLBR SK678 16 . The black circle highlights the surface adjacent to C11/OH11 and shows a more defined binding pocket in the characterized Neu5Ac-selective SLBRs. Molecular Dynamics (MD) Simulations of SLBR Hsa and Glycan Motions To calculate whether motions in SLBR Hsa might affect interactions with Neu5Ac- or Neu5Gc-terminated sialoglycans, we conducted molecular dynamics (MD) simulations. In these simulations, we separately calculated SLBR Hsa flexibility and sialyl disaccharide flexibility. The coordinates for Neu5Gcα2-3Gal did not include the methyl aglycon, so both sialoglycans had equivalent structures. Simulations of SLBR Hsa flexibility were initiated from the unliganded conformation of SLBR Hsa 16 , allowing the loops to equilibrate around each ligand without pre-imposed bias. As in prior simulations 16 , the CD-, EF-, and FG-loops adjacent to the binding pocket exhibited the greatest flexibility, supported by both root-mean-square fluctuation (RMSF) ( Supplementary Fig. 3 ) and crystallographic temperature factor analyses ( Supplementary Fig. 4 ). In addition, the EF loop of SLBR Hsa closed over both Neu5Acα2-3Gal and Neu5Gcα2-3Gal, allowing the SLBR Hsa K335 backbone carbonyl to approach the O4 atom of each sialoglycan ( Supplementary Fig. 5 ). This differs from what was observed in the X-ray crystal structures, in which only the SLBR Hsa –Neu5Gc structure had a closed loop ( Supplementary Fig. 1A ). We particularly evaluated SLBR Hsa V370 on the G strand, which is analogous to the SLBR SrpA Y368 that hydrogen-bonds with Neu5Gc OH11. SLBR Hsa V370 did not exhibit flexibility or approach either sialoglycan. In fact, no protein atoms came within 3 Å of the Neu5Ac/Neu5Gc C11 or Neu5Gc OH11 at any point during the simulation. These predictions suggest that, on the timescale of the simulation, motions in SLBR Hsa do not promote stable direct contacts with C11 or OH11. We designed a second set of calculations to evaluate whether sialoglycan flexibility might contribute to transient interactions with SLBR Hsa by allowing C11, OH11, or other atoms to approach the protein surface ( Supplementary Fig. 6 ). These simulations were initiated with the EF loop of SLBR Hsa in the closed conformation around each disaccharide. Throughout the simulations, the Neu5Acα2-3Gal and Neu5Gcα2-3Gal disaccharides remained stably bound with a conserved orientation. The sialic acid and galactose moieties showed minimal positional fluctuation. In the Neu5Gc-bound simulation, the OH11 did not form persistent contacts with the protein surface. These findings are consistent with the crystal structure of SLBR Hsa –Neu5Gc, in which OH11 is rotated away from the protein and SLBR Hsa does not directly engage Neu5Gc OH11 ( Fig. 3 ). The resulting probability distributions in the replicates showed show a narrow probability distribution around the pose of the crystal structure ( Supplementary Fig. 6 ). The mean RMSD is 0.85 Å ± 0.14 Å for Neu5Ac and 0.83 Å ± 0.15 Å for Neu5Gc. Binding of SLBR Hsa V170 and SLBR SrpA Y368 Mutants to Purified Glycans Synthetic Disaccharides We next explored the potential for indirect influences that might help SLBRs distinguish between these two forms of sialic acid. We focused on residues analogous to SLBR SrpA Y368 , whose side chain hydroxyl forms a hydrogen bond with the OH11 group of Neu5Gc ( Fig. 3B ). The Cα of SLBR SrpA Y368 is 7.6 Å from OH11 and the close approach of the side chain hydroxyl is due to the length of the residue ( Fig. 3B ). SLBR SrpA Y368 corresponds with SLBR Hsa V370 ( Fig. 3A, 3B ). While SLBR SrpA Y368 makes a hydrogen bond to the Neu5Gc OH11 ( Fig. 3B ), other amino acids at this position are not capable of a hydrogen-bonding interaction. Among characterized SLBRs, residues at the equivalent position are typically hydrophobic: Val, Ile, Tyr, or Phe ( Fig. 5 ). These residues are also smaller than Tyr. For example, the closest side chain atoms from the shorter SLBR Hsa V370 7.5 Å away from C11 ( Fig. 3A , Supplementary Fig. 2A ). Finally, except for SLBR SrpA Y368 , these analogous residues do not contact the ligand. For example, SLBR Hsa S336 and SLBR Hsa Y338 are located between the SLBR Hsa V370 side chain and C11, blocking the possibility of direct contact ( Fig. 3A ). Download figure Open in new tab Figure 5 Structure-based sequence alignment of SLBRs with reported Neu5Ac/Neu5Gc binding data Sequences are from WP_081102781.1 from S. gordonii strain Challis (SLBR Hsa ) 16 , 61 , WP_045635027.1 from S. gordonii strain UB10712 16 , 62 , WP_011836739.1 from S. sanguinis strain SK36 (SLBR SrpA ) 32 , 63 , WP_080555651.1 from S. sanguinis strain SK1 (SLBR SK1a and SLBR SK1b ) 34 , 64 , WP_125444035.1 from S. sanguinis strain SK678 16 , 64 , and WP_125444382.1 from S. gordonii strain M99 (SLBR GspB ) 14 , 65 . The highlighted residue is mutated in this study. To investigate whether residues at this position play an allosteric role in distinguishing between sialic acid forms, we made substitutions in SLBR Hsa and SLBR SrpA . SLBR Hsa V370 was mutated to Ile, Phe, or Tyr and SLBR SrpA Y368 was mutated to Val, Ile, or Phe, so that each of these SLBR scaffolds had a version with Val, Ile, Tyr, or Phe at this position. We assessed binding to purified and biotinylated Neu5Acα2-3Gal or Neu5Gcα2-3Gal by ELISA ( Fig. 6 , 7 ), evaluating both the overall level of binding and the preference for Neu5Ac-versus Neu5Gc-terminated disaccharides. Consistent with previous reports, wild-type SLBR Hsa bound sialyl disaccharides at much higher levels than SLBR SrpA , but each wild-type SLBR had no preference for sialyl disaccharides terminating in Neu5Ac or Neu5Gc ( Fig. 6 , 7 ). Download figure Open in new tab Figure 6 Wildtype and mutant SLBR Hsa binding to purified biotinylated disaccharides A) ELISA curves for wildtype and mutant GST-tagged SLBR Hsa (GST-SLBR Hsa ) binding to biotinylated Neu5Ac- and Neu5Gc-terminated disaccharides at the indicated concentrations. Measurements were performed using 500 nM of immobilized GST-SLBR Hsa , and the indicated concentrations of each ligand are shown as the mean ± SD. (n = 3 independent experiments performed on protein from a single preparation). B) Comparison of the levels of binding of wildtype and mutant GST-SLBR Hsa at a concentration of 1 μg/mL biotinylated disaccharides. p-values for Neu5Ac versus Neu5Gc binding to SLBR Hsa are: SLBR Hsa WT p= 0.9585, SLBR Hsa V370F p= 0.0121, SLBR Hsa V370I p= <0.0001, SLBR Hsa V370Y p= <0.0001, as evaluated by one-way ANOVA, C) Preference of wildtype or mutant GST-SLBR Hsa for Neu5Ac ( purple ) versus Neu5Gc ( blue ). Percentages were calculated by dividing measured A 450 values corresponding to either Neu5Ac or Neu5Gc by the additive absorbance value (Neu5Ac+Neu5Gc). As compared to wild-type SLBR Hsa , SLBR Hsa V370I and SLBR Hsa V370Y exhibited reduced but statistically significant Neu5Acα2-3Gal binding, while SLBR Hsa V370Y lacked statistically significant binding under the conditions tested ( Fig. 6A, 6B ). In contrast, all SLBR Hsa proteins retained binding to Neu5Gcα2-3Gal, although overall levels were moderately reduced ( Fig. 6A, 6B ). Because of the disproportionate loss of Neu5Acα2-3Gal binding, all SLBR Hsa mutants became more selective for Neu5Gc-terminated disaccharides ( Fig. 6C ). Mutations in SLBR SrpA produced an even more striking result. All variants showed substantially increased relative binding to Neu5Gc-termined disaccharides when compared to the wild-type SLBR SrpA . The strongest effect was observed with the SLBR SrpA Y368I , where binding to Neu5Gcα2-3Gal increased 5.1-fold when 1 µg/mL of each biotinylated disaccharide was used ( Fig. 7A, 7B ). The SLBR SrpA Y368V (3.9-fold increase) and SLBR SrpA Y368F (3.9-fold increase) mutants also showed substantial increases in Neu5Gcα2-3Gal binding ( Fig. 7A, 7B ). At the same time, Neu5Acα2-3Gal binding increased 2.7-fold in SLBR SrpA Y368F but was statistically identical in the remaining mutants. Because of the very large gains in Neu5Gcα2-3Gal binding, SLBR SrpA mutants were even more strongly selective for Neu5Gc-terminated disaccharides than were SLBR Hsa mutants ( Fig. 7C ). Download figure Open in new tab Figure 7 Wildtype and mutant SLBR SrpA binding to purified and biotinylated disaccharides A) ELISA curves for wildtype and mutant GST-tagged SLBR Hsa (GST-SLBR Hsa ) binding to biotinylated Neu5Ac- and Neu5Gc-terminated disaccharides at the indicated concentrations. Measurements were performed using 500 nM of immobilized GST-SLBR, and the indicated concentrations of each ligand are shown as the mean ± SD. (n = 3 independent experiments performed on protein from a single preparation). B) Comparison of the levels of wildtype and mutant GST-SLBR SrpA binding to 1 μg/mL biotinylated Neu5Ac versus Neu5Gc disaccharides. p-values for Neu5Ac versus Neu5Gc binding to SLBR SrpA are: SLBR SrpA WT p= 0.9407, SLBR SrpA Y368F p= <0.0001, SLBR SrpA Y368I p= <0.0001, SLBR SrpA Y368V p= <0.0001, as evaluated by one-way ANOVA, C) Preference of wildtype or mutant GST-SLBR SrpA for Neu5Ac ( purple ) versus Neu5Gc ( blue ). Percentages were calculated by dividing measured A 450 values corresponding to either Neu5Ac or Neu5Gc by the additive absorbance value (Neu5Ac+Neu5Gc). Taken together, our mutational analysis identified that the SLBR Hsa V370 and SLBR SrpA Y368 positions affect sialic acid selectivity and that all tested mutations at this position increased Neu5Gc selectivity. The substantial gains in Neu5Gc preference were particularly intriguing because the substitutions maintain hydrophobicity near the sialoglycan binding site, and Neu5Gc is more hydrophilic. Far Western Analysis with Wild-type and Neu5Gc-selective mutants We next investigated binding to authentic glycoproteins from human and rat sources by far Western analysis ( Fig. 8 , 9 ). Within the limits of detection, human plasma contains glycoproteins that only terminate in Neu5Ac and its derivatives, while rats have glycoproteins terminating in both Neu5Ac and Neu5Gc 23 . In addition, this evaluates how selectivity for Neu5Ac versus Neu5Gc is combined with potential contributions from glycan branching or protein context. Download figure Open in new tab Figure 8 SLBR Hsa binding to human or rat plasma glycoproteins A) Far-Western blot of wild-type and mutant GST-SLBR Hsa against plasma glycoproteins. Glycoproteins were separated by electrophoresis through a 3–8% polyacrylamide gradient, and then stained. No signals were detected outside of the cropped region. As previously identified 6 , the proteins highlighted by the red box are human GPIbα (149 kDa, purple arrow ) or rat GPIbα (160 kDa, pink arrow ). B) Dosimetry of blots showing the total amount of binding to all proteins, without regard to molecular weight, measured with IMAGEJ version 1.54 66 . p-values for human versus rat binding to SLBR Hsa are: SLBR Hsa WT p= <0.0001, SLBR Hsa V370F p= 0.0012, SLBR Hsa V370I p= 0.0155, SLBR Hsa V370Y p= 0.0214, as evaluated by two-way ANOVA, C) Preference of wildtype or mutant GST-SLBR Hsa for human (Neu5Ac only, purple ) versus rat (Neu5Ac/Neu5Gc, pink ). Percentages were calculated by dividing pixel counts corresponding to either human or rat samples by the additive pixel counts (human + rat). Contrary to what was observed in ELISAs with synthetic sialoglycans ( Fig. 6 ), wild-type SLBR Hsa showed a robust interaction with the human plasma glycoproteins ( Fig. 8A, 8B ) and a substantial preference for binding to glycoproteins within human plasma ( Fig. 8C ). However, there are some curious nuances. Of particular note is binding to the ∼150 kDa GPIbα glycoprotein implicated as the receptor in endocardial infection ( Fig. 8A ). SLBR Hsa exhibits more total binding to human plasma glycoproteins than rat plasma glycoproteins ( Fig. 8B ), but much of this is off target with respect to GPIbα ( Fig. 8A ). While there is less total binding of SLBR Hsa to rat plasma glycoproteins, this SLBR more selectively recognizes rat GPIbα ( Fig. 8A ). Among SLBR Hsa mutants, SLBR Hsa V370F retained a preference for human plasma glycoproteins, albeit at a 3-fold decrease in total binding ( Fig. 8A, 8B ). SLBR Hsa V370F also lost detectable binding to human GPIbα and only bound to off-target glycoproteins not associated with endocarditis ( Fig. 8A ). All detectable binding of SLBR Hsa V370F to rat plasma glycoproteins is to GPIbα ( Fig. 8A ). The remaining two SLBR Hsa mutants only detectably bound to rat GPIbα ( Fig. 8A ). Wild-type SLBR SrpA bound more robustly to rat plasma ( Fig. 9A, 9B ), again in contrast to the statistically identical binding to Neu5Ac- and Neu5Gc-terminated synthetic disaccharides in ELISA ( Fig. 7 ). Moreover, almost all detectable SLBR SrpA binding was to GPIbα in both human and rat plasma ( Fig. 9A ). Of the SLBR SrpA mutants, SLBR SrpA Y368V and SLBR SrpA Y368I either had statistically identical or modestly increased the binding to binding to rat GPIbα ( Fig. 9A, 9B ), which contrasted with the large increase of binding of each of these SLBR mutants to purified Neu5Gc-terminated disaccharides ( Fig. 7 ). For example, wild-type SLBR SrpA and SLBR SrpA Y368F had statistically identical binding to rat GPIbα ( Fig. 9A, 9B ), even though SLBR SrpA Y368F had 5-fold greater binding to synthetic Neu5Gc-terminated disaccharides in ELISA ( Fig. 7A, 7B ). The SLBR SrpA Y368F mutant showed barely detectable binding to human plasma glycoproteins, despite a ∼3-fold increase in binding to Neu5Acα2-3Gal binding in the ELISA assays ( Fig. 9 ). The remaining two SLBR SrpA mutants had undetectable binding to human plasma glycoproteins under these conditions, and all SLBR SrpA mutants substantially shifted the binding preference toward rat glycoproteins ( Fig. 9C ). Download figure Open in new tab Figure 9 Far Western analysis of SLBR SrpA binding to human or rat plasma A) Far-Western blot of wild-type and mutant GST-SLBR SrpA against plasma glycoproteins. Glycoproteins were separated by electrophoresis through a 3–8% polyacrylamide gradient, and then stained. No signals were detected outside of the cropped region. The proteins in the red box are human GPIbα (149 kDa, purple arrows ) or rat (160 kDa, pink arrows ) GPIbα 6 . B) Dosimetry of blots showing the total amount of binding to all proteins, without regard to molecular weight, measured with IMAGEJ version 1.54 66 . p-values for human versus rat binding to SLBR SrpA are: SLBR SrpA WT p= 0.0011, SLBR SrpA Y368F p= 0.0002, SLBR SrpA Y368I p= <0.0001, SLBR SrpA Y368V p= <0.0001, as evaluated by two-way ANOVA, C) Preference of wildtype or mutant GST-SLBR Hsa for human (Neu5Ac only, purple ) versus rat (Neu5Ac/Neu5Gc, pink ). Percentages were calculated by dividing pixel counts corresponding to either human or rat samples by the additive pixel counts (human + rat). Discussion Our data provide insight into how SLBRs from viridans group streptococci engage Neu5Ac- and Neu5Gc-terminated sialoglycans ( Fig. 1 ). We define the structural basis for binding of SLBR Hsa to the Neu5Gcα2-3Gal and Neu5Gcα2-3Gal sialyl disaccharides ( Fig. 2 - 4 , Supplementary Fig 1 , 2 ), show that motions are unlikely to affect Neu5Ac/Neu5Gc selectivity ( Supplementary Fig 3-6), and demonstrate that Neu5Ac/Neu5Gc preference can be modified through mutation of an allosteric site ( Fig. 5 - 7 ). Furthermore, we show that that binding to synthetic Neu5Ac- and Neu5Gc-terminated disaccharides only partially predicts binding to authentic human and rat plasma sialoglycoproteins ( Fig. 6 - 9 ). Intriguingly, SLBR mutants also showed narrowed glycoprotein engagement ( Fig. 8 , 9 ), losing off-target binding and very selectively engaging the GPIbα glycoprotein that is implicated as the receptor for infective endocarditis 7 , 35 , 36 . Several aspects of these results suggest important nuances in how SLBRs mediate host glycoprotein recognition. A key finding was that Neu5Ac/Neu5Gc selectivity appears to be modulated by allosteric effects rather than by direct contacts ( Fig. 2 , 3 ). In addition, there was no evidence that flexibility contributes meaningfully to Neu5Ac/Neu5Gc cross-reactivity ( Supplementary Fig. 3-6 ). This fully contrasts with how SLBRs distinguish between tri- and tetrasaccharide sialoglycans with different chemical compositions, in which binding preferences are largely dictated by direct SLBR-ligand interactions, as assisted by protein flexibility 16 . In those cases, chimeragenesis and point mutagenesis resulted in predictable changes to the sialoglycan binding repertoire 16 and flexibility of the CD-, EF-, and FG-loops ( Fig. 2A ) were shown to contribute to broad selectivity 16 . Another striking finding was the consistent gain in Neu5Gc preference for both SLBRs following point mutagenesis ( Fig. 6 - 9 ). This asymmetry may arise from differences in the requirements for each form of sialic to interact with SLBRs ( Fig. 2 - 4 ). A polar OH11 of Neu5Gc may benefit from a more open binding pocket ( Fig. 4A - 4C ) to allow local motions and enhanced solvent exposure. This may be recapitulated by perturbed local packing that inevitably accompanies mutagenesis. A hydrophobic Neu5Ac C11 methyl group may require more precise packing and desolvation ( Fig. 4D - 4F ). Despite this, all naturally occurring SLBRs that have been experimentally characterized either exhibit a strong binding preference toward sialoglycans terminating in Neu5Ac 2 , 16 , 19 or bind equivalently to sialoglycans terminating in Neu5Ac or Neu5Gc 2 ( Fig. 6A , 7A ). While it is not clear why this is, one possibility is that the absence of characterized SLBRs that prefer Neu5Gc-terminated sialoglycans may reflect that the library of available viridans group streptococci favors bacteria isolated from humans 2 – 4 . These isolates face selective pressure to engage Neu5Ac-terminated sialoglycosides of humans 5 , 6 . The imperfect correlation between Neu5Ac/Neu5Gc selectivity ( Fig. 6 , 7 ) and host preference ( Fig. 8 , 9 ) extends our understanding of host tropism by sialoglycan-binding pathogens. One hypothesis for the divergence in sialic acid composition between humans and non-human animals involves the evolutionary response to host-pathogen interplay. Non-human animals synthesize CMP-linked Neu5Gc when CMP-Neu5Ac hydroxylase enzyme hydroxylates the N -acetyl group (i.e. C11) of CMP-Neu5Ac. Our study used samples from rats, where Neu5Gc levels have been experimentally measured at different levels depending on the biological location. The ratio in developing lungs measured as ∼75% Neu5Ac and ∼25% Neu5Gc 23 while the level in rat GPIbα extract is measured as ∼28% Neu5Ac and ∼58% Neu5Gc 6 . Humans have an inactive CMP-Neu5Ac hydroxylase enzyme due to a loss-of-function mutation occurring approximately two million years ago 21 , 25 , 37 , 38 , and therefore only synthesize Neu5Ac. It has been postulated that this human loss of function mutation conferred protection against ancestral forms of Plasmodium , the parasitic protozoan responsible for malaria 39 as some modern P. falciparum strains rely primarily on sialic acid for invasion 40 , 41 . These results support a model where the relationship between pathogenesis and sialic acid identity is not straightforward. Instead linkage specificity (e.g., α2-3 vs α2-6 of C6 to the underlying Gal) may play a large role than sialic acid identity in determining virulence. This aligns well with other characterized pathogens that bind to sialoglycans. One example is observed in the influenza virus, which binds to host sialoglycans through hemagglutinins. The H1 and H3 hemagglutinins specifically bind α2-6 linked sialic acids, which are abundant in mammalian airways 42 . These strains are infectious to humans and other mammals. By contrast, H5 hemagglutinin found in strains of the avian flu binds to α2-3 linked sialic acids, which are abundant in avian airways but rare in mammalian airways 43 , 44 . The H5N1 avian flu has a high mortality rate in both mammals and birds, but H5N1 strains are substantially less infective for humans at the present time because of this linkage selectivity 45 . Intriguingly, Neu5Gc-terminated sialoglycans can act as decoy receptors for some strains of influenza, where they support hemagglutinin binding but inhibit viral entry 35 – 37 . Coronaviruses such as SARS-CoV and SARS-CoV2 can also engage host sialoglycans 46 – 48 , and this interaction synergizes with binding to ACE2 to promote viral entry 46 – 48 . Although this finding is quite recent, early work similarly suggests narrow linkage selectivity for this interaction 46 – 48 . Our results suggest that host tropism of viridans group streptococci blends Neu5Ac/Neu5Gc selectivity with broader glycan context. Host glycoprotein engagement is certainly influenced by the ability to bind Neu5Ac versus Neu5Gc-capped sialoglycans, as evidenced by shifts in SLBR binding toward rat glycoproteins as Neu5Gc preference increased ( Fig. 6 - 9 ). However, SLBR interactions with synthetic sialyl disaccharides did not fully predict binding to authentic plasma glycoproteins, which can have larger, more complex underlying glycan structures. This highlights a secondary role for sialic acid identity relative to other determinants, such as glycan linkage and glycoprotein presentation. The discrepancy between plasma glycoprotein binding and synthetic sialyl disaccharide recognition is unlikely to result from alternative glycoprotein engagement. The only other sialic acid derivative present on human cells is Kdn, which accounts for less than 1% of surface glycans and is rarely incorporated into glycoproteins 49 . Additionally, we are not aware of any literature suggesting that SLBRs could engage non-sialoglycoside glycoproteins. Together, our data support a model in which Neu5Ac/Neu5Gc preference modulates, but does not uniquely determine, SLBR-mediated host glycoprotein recognition by viridans group streptococci. By uncovering distinct structural and allosteric mechanisms of sialoglycan recognition, this work extends our understanding of SLBR specificity and host range, offering insight into bacterial adaptation. Finally, our work identifies that models of host tropism will benefit from a nuanced view of glycoprotein chemistry that encompasses sialic acid identity, glycan context, and presentation. Experimental Procedures Protein Expression and Purification for X-ray Crystallography SLBR Hsa was expressed and purified as previously described 16 . Briefly, the pSV278 vector (Vanderbilt university) appends a His-maltose-binding protein (MBP) tag at the N-terminus of SLBR Hsa, followed by a thrombin cleavage site. His 6 -MBP-SLBR Hsa was expressed in Escherichia coli BL21 (DE3) in Terrific Broth medium supplemented with 50 µg/ml kanamycin at 37°C. At an OD 600 of 1.0, the temperature was lowered to 24°C, and expression was induced with 1 mM isopropyl β-d-thiogalactopyranoside (IPTG) for 6 hrs. Cells were harvested by centrifugation at 5,000 g for 15 min, washed with 0.1 M Tris-HCl, pH 7.5, and stored at −20°C before purification. Frozen cells were resuspended in buffer containing 20 – 50 mM Tris-HCl, pH 7.5, 150 – 200 mM NaCl, 1 mM EDTA, 1 mM PMSF, 2 µg/ml Leupeptin, 2 µg/ml Pepstatin, then disrupted by sonication. Lysate was clarified by centrifugation at 38,500 g for 35 – 60 min and passed through a 0.45 µm filter. Purification of His 6 -MBP-SLBR Hsa was performed at 4°C with an MBP-Trap column and eluted in 10 mM maltose. Eluted proteins were concentrated in a 10 kDa molecular weight cutoff concentrator and exchanged into buffer containing 20 mM Tris-HCl, pH 7.5, and 200 mM NaCl. The His 6 -MBP affinity tags were cleaved from SLBR Hsa with 1 U of thrombin per mg of His 6 -MBP-SLBR Hsa overnight at 4°C. The cleaved His 6 -MBP tag was separated from pure SLBR Hsa using a Superdex 200 Increase 10/30 GL column equilibrated in 20 mM Tris-HCl, pH 7.5, and 200 mM NaCl. After purification, the protein was > 95% pure, as assessed by SDS-PAGE, and was stored at −80°C. Crystallization and Collection of X-ray Diffraction Data Crystals of SLBR Hsa (21.6 mg/ml in 20 mM Tris-HCl, pH 7.2) were formed by the sitting drop vapor diffusion method by equilibrating 1 µL protein and 2 µL reservoir solution over 50 µL of reservoir solution (0.1 M Succinate/Phosphate/Glycine pH 10.0 and 25% PEG 3350). Co-crystals of SLBR Hsa with sialoglycan ligands were prepared by soaking fully formed crystals in reservoir solution supplemented with 5 mM of each ligand for 20 hr. Crystals did not require cryoprotection beyond the reservoir solution and were cryocooled by plunging into liquid nitrogen. Data were collected at −180°C on beamline 9-2 at the Stanford Synchrotron Radiation Lightsource. Data were processed in HKL2000 50 . Structure Determination and Refinement The structure of each sialoglycan-bound SLBR Hsa was determined using isomorphous replacement by removing all solvent molecules from unliganded SLBR Hsa (PDB entry 6EFC 16 ) and performing rigid body refinement in PHENIX 51 . The resultant model was improved using alternate rounds of model building in COOT 52 and refinement in PHENIX 51 . Throughout the process, the R free reflections were selected to be the same as for the unliganded SLBR Hsa (PDB entry 6EFC 16 ). Data collection and refinement statistics are listed in Table 1 . Sialoglycan reagents Neu5Acα2-3Gal and Neu5Gcα2-3GalβOMe used in crystallography studies were prepared as previously reported 32 , 33 . Biotinylated sialyl disaccharides for the ELISAs were purchased from Sigma (GNZ-0035-BM for Neu5Ac and GNZ-0018-BM for Neu5Gc). MD Simulations The crystal structures of SLBR Hsa bound to either Neu5Acα2-3Gal or Neu5Gcα2-3Gal were used to generate starting models for MD simulations. MD was performed on the proteins and glycan ligands using the Amber14 ff14SB 53 and Glycam06 54 force fields, respectively, with a non-bonded cutoff of 10 Å using the Particle Mesh Ewald algorithm 55 . Each protein-glycan system was hydrated by water model TIP3P 56 using an octahedral box of 10 Å around the protein in each direction. Initially, the protein was held fixed with a force constant of 500 kcal mol -1 Å -2 while the system was energy minimized with 500 steps of steepest descent. This was followed by 500 steps of energy minimization with the conjugate gradient method. In a second minimization step, the restraints on the protein were removed, and 1000 steps of steepest descent minimization were performed, followed by 1500 steps of conjugate gradient. The system was heated to 300 K while holding the protein fixed with a force constant of 10 kcal mol-1 Å-2 for 1000 steps. Then, the restraints were removed, and 1000 MD steps were performed. The SHAKE algorithm 57 was used to constrain all bonds involving hydrogen in the simulations. MD production runs were performed at 300 K using the NPT ensemble and a 2-fs time step. The temperature was fixed with the Langevin dynamics thermostat 58 , and the pressure was fixed with the Monte Carlo barostat 59 . Three independent runs were performed for each simulation. All analyses were done using the Pytraj package 60 . Protein Expression and Purification for ELISAs and Far Western Blotting SLBR Hsa , SLBR SrpA , and all variants were expressed and purified as previously described 16 . Point mutants were created from already cloned SLBRs in vector pBG101 (Vanderbilt University), which encodes a His 6 -Glutathione-S-transferase (GST) tag at the N-terminus, followed by a 3C protease cleavage site. His 6 -GST-SLBRs were expressed in Escherichia coli BL21 (DE3) in Miller’s Luria Broth at 37°C with 50ug/mL of kanamycin for ∼3 hr to reach an A 600 of 0.80. SLBR expression was induced with 1 mM IPTG for 3 hours at 24°C. Cells were harvested by centrifugation at 7,000 x g for 20 minutes. Cell pellets were resuspended in 125 mM Tris, 150 mM sodium chloride, pH 8.0, supplemented with 1 mM EDTA, 1 mM PMSF, 2 µg/ml Leupeptin, 2 µg/ml Pepstatin, then lysed by sonication. Lysate was clarified by centrifugation at 18,000 x g for 1 hour. The supernatant was filtered (0.45µm) and purified using glutathione-sepharose as instructed by the manufacturer (Thermo, 16108). After purification, proteins were concentrated in a 30 kDa molecular weight cutoff concentrator and buffer exchanged using a Superdex 200 Increase 10/30 GL column equilibrated in 1X Dulbecco′s Phosphate Buffered Saline (DPBS) with calcium and magnesium (Sigma, D1283). The protein was > 95% pure, as assessed by SDS-PAGE, and was stored at −80°C. ELISAs The binding of biotinylated sialoglycans to immobilized GST-SLBRs was performed as described 2 , 7 , 16 . In short, purified SLBRs were diluted to 500 µM in DPBS and added to a 96-well microtiter plate. Plates were incubated overnight at 4°C. Unbound proteins were removed by aspiration, and wells were rinsed with DPBS. Biotinylated glycans were diluted to the indicated concentrations in DPBS containing 1X Blocking Reagent (Roche, 11585762001) and incubated for 1 hr at room temperature. Wells were rinsed three times with DPBS. Streptavidin-conjugated horseradish peroxidase (Sigma, S5512) was added to each well, and the plate was incubated for 1 hr at room temperature. The wells were washed twice with DPBS, and then a solution of 0.4 mg o-Phenylenediamine dihydrochloride (Sigma, P8787) per mL phosphate-citrate buffer (Sigma, P4922) was added to the wells. The absorbance at 450 nm was measured after approximately 20 min. Data were plotted as the means ± standard deviations, with n = 3. Far-Western Blotting Far western blotting was previously described 2 , 7 , 16 . Briefly, human or rat plasma (Innovative Research) was diluted 1:10 into 10 mM Tris, 1 mM EDTA, pH 8, combined with LDS sample buffer and DTT (50 mM final concentration). Samples were boiled for 10 min, and proteins were separated by electrophoresis on 3–8% polyacrylamide gradient gels (Life Technologies), and then transferred to BioTraceNT (Pall Corporation). Membranes were incubated for 1 h at room temperature with 1× Blocking Reagent in DPBS. GST-SLBRs were then added to a final concentration of 5 nM, and the membranes were incubated for 90 min at room temperature with gentle rocking. After rinsing three times with DPBS, the membranes were incubated for 1 h at room temperature with anti-GST diluted 1:5000 in DPBS containing 1× Blocking Reagent. Membranes were rinsed three times with DPBS and then incubated for 1 h at room temperature with horseradish peroxidase-conjugated goat anti-rabbit antibodies diluted 1:50,000 in DPBS. Membranes were again rinsed three times with DPBS and then developed with SuperSignal West Pico (Thermo Scientific). Acknowledgements We thank L. Loukachevitch for experimental assistance in the early stages of this work. This work was supported by NIH grant GM137458 to TMI, BAB, XC, JCS, and DE019807 to SR, TMI. KMM was supported by NIH training grant GM007628. HES was supported by the NIH Training grant EY007135. Use of the Stanford Synchrotron Radiation Lightsource, SLAC National Accelerator Laboratory, is supported by the U.S. Department of Energy, Office of Science, Office of Basic Energy Sciences under Contract No. DE-AC02-76SF00515. The SSRL Structural Molecular Biology Program is supported by the DOE Office of Biological and Environmental Research, and by the National Institutes of Health, National Institute of General Medical Sciences (P30GM133894). 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