Human beta1-coronavirus (β1CoV) OC43 emerged relatively recently through a single zoonotic introduction. Like related animal β1CoVs, OC43 uses 9-O-acetylated sialic acid as receptor determinant. β1CoV receptor binding is typically controlled by attachment/fusion spike protein S and receptor-binding/receptor-destroying hemagglutinin-esterase protein HE. We show that following OC43's introduction into humans, HE-mediated receptor binding was selected against and ultimately lost through progressive accumulation of mutations in the HE lectin domain. Consequently, virion-associated receptor-destroying activity toward multivalent glycoconjugates was reduced and altered such that some clustered receptor populations are no longer cleaved. Loss of HE lectin function was also observed for another respiratory human coronavirus, HKU1. This thus appears to be an adaptation to the sialoglycome of the human respiratory tract and for replication in human airways. The findings suggest that the dynamics of virion-glycan interactions contribute to host tropism. Our observations are relevant also to other human respiratory viruses of zoonotic origin, particularly influenza A virus.

Toll-like receptors (TLRs) are crucial in innate recognition of invading micro-organisms and their subsequent clearance. Bacteria are not passive bystanders and have evolved complex evasion mechanisms. Staphylococcus aureus secretes a potent TLR2 antagonist, staphylococcal superantigen-like protein 3 (SSL3), which prevents receptor stimulation by pathogen-associated lipopeptides. Here, we present crystal structures of SSL3 and its complex with TLR2. The structure reveals that formation of the specific inhibitory complex is predominantly mediated by hydrophobic contacts between SSL3 and TLR2 and does not involve interaction of TLR2-glycans with the conserved Lewis X binding site of SSL3. In the complex, SSL3 partially covers the entrance to the lipopeptide binding pocket in TLR2, reducing its size by ∼50%. We show that this is sufficient to inhibit binding of agonist Pam 2 CSK 4 effectively, yet allows SSL3 to bind to an already formed TLR2-Pam 2 CSK 4 complex. The binding site of SSL3 overlaps those of TLR2 dimerization partners TLR1 and TLR6 extensively. Combined, our data reveal a robust dual mechanism in which SSL3 interferes with TLR2 activation at two stages: by binding to TLR2, it blocks ligand binding and thus inhibits activation. Second, by interacting with an already formed TLR2-lipopeptide complex, it prevents TLR heterodimerization and downstream signaling.S. aureus | Toll-like receptor | immune evasion | innate immunity | crystal structure

Hemagglutinin-esterases (HEs) are bimodular envelope proteins of orthomyxoviruses, toroviruses, and coronaviruses with a carbohydrate-binding "lectin" domain appended to a receptordestroying sialate-O-acetylesterase ("esterase"). In concert, these domains facilitate dynamic virion attachment to cell-surface sialoglycans. Most HEs (type I) target 9-O-acetylated sialic acids (9-O-Ac-Sias), but one group of coronaviruses switched to using 4-O-Ac-Sias instead (type II). This specificity shift required quasisynchronous adaptations in the Sia-binding sites of both lectin and esterase domains. Previously, a partially disordered crystal structure of a type II HE revealed how the shift in lectin ligand specificity was achieved. How the switch in esterase substrate specificity was realized remained unresolved, however. Here, we present a complete structure of a type II HE with a receptor analog in the catalytic site and identify the mutations underlying the 9-O-to 4-O-Ac-Sia substrate switch. We show that (i) common principles pertaining to the stereochemistry of proteincarbohydrate interactions were at the core of the transition in lectin ligand and esterase substrate specificity; (ii) in consequence, the switch in O-Ac-Sia specificity could be readily accomplished via convergent intramolecular coevolution with only modest architectural changes in lectin and esterase domains; and (iii) a single, inconspicuous Ala-to-Ser substitution in the catalytic site was key to the emergence of the type II HEs. Our findings provide fundamental insights into how proteins "see" sugars and how this affects protein and virus evolution.A mong host cell surface determinants for pathogen adherence, sialic acids (Sias) rank prominently (1, 2). Representatives of at least 11 families of vertebrate viruses use Sia as primary entry receptor and/or attachment factor (3, 4). Viral adherence to sialoglycans, however, comes with inherent complexities related to (i) the sheer ubiquity of receptor determinants that may act as "decoys" when present on off-target cells and non-cell-associated glycoconjugates, and (ii) the dense clustering that is characteristic to glycotopes and that may augment the apparent affinity of ligandlectin interactions by orders of magnitude (5, 6). Viruses may avoid inadvertent virion binding to nonproductive sites by being selective for particular sialoglycan subtypes so that attachment is dependent on Sia linkage type, the underlying glycan chain, and/or the absence or presence of specific postsynthetic Sia modifications (2, 7, 8). Moreover, as an apparent strategy to evade irremediable binding to decoy receptors, viral sialolectins typically are of low affinity, with dissociation constants in the millimolar range (reviewed in ref.3). In consequence, virion-Sia interactions are intrinsically dynamic and the affinity of the virolectins would appear to be fine-tuned such as to ensure reversibility of virion attachment. In most viruses, reversibility is exclusively subject to the lectin-ligand binding equilibrium. Some, howev...

Glycoprotein VI (GPVI) mediates collagen-induced platelet activation after vascular damage, and is an important contributor to the onset of thrombosis, heart attack and stroke. Animal models of thrombosis have identified GPVI as a promising target for antithrombotic therapy. Although for many years the crystal structure of GPVI has been known, the essential details of its interaction with collagen have remained elusive. Here, we present crystal structures of the GPVI ectodomain bound to triple-helical collagen peptides, which reveal a collagen-binding site across the β-sheet of the D1-domain. Mutagenesis and binding studies confirm the observed binding site, and identify Trp76, Arg38 and Glu40 as essential residues for binding to fibrillar collagens and collagen-related peptide (CRP). GPVI binds a site on collagen comprising two collagen chains with the core formed by the sequence motif OGPOGP. Potent GPVI-binding peptides from Toolkit-III all contain OGPOGP, weaker binding peptides frequently contain a partial motif varying at either terminus. Alanine-scanning of peptide III-30 also identified two AGPOGP motifs that contribute to GPVI-binding, but steric hindrance between GPVI-molecules restricts the maximum binding capacity. We further show that no cooperative interactions could occur between two GPVI-monomers binding to a stretch of (GPO)5, and that binding of two or more GPVI-molecules to a fibril-embedded helix requires non-overlapping OGPOGP-motifs. Our structure confirms the previously suggested similarity in collagen binding between GPVI and Leukocyte-Associated Immunoglobulin-like Receptor 1 (LAIR-1), but also indicates significant differences that may be exploited for the development of receptor-specific therapeutics.

Complement factor C5a is one of the most powerful pro-inflammatory agents involved in recruitment of leukocytes, activation of phagocytes and other inflammatory responses. C5a triggers inflammatory responses by binding to its G-protein-coupled C5a-receptor (C5aR). Excessive or erroneous activation of the C5aR has been implicated in numerous inflammatory diseases. The C5aR is therefore a key target in the development of specific anti-inflammatory compounds. A very potent natural inhibitor of the C5aR is the 121-residue chemotaxis inhibitory protein of Staphylococcus aureus (CHIPS). Although CHIPS effectively blocks C5aR activation by binding tightly to its extra-cellular N terminus, it is not suitable as a potential anti-inflammatory drug due to its immunogenic properties. As a first step in the development of an improved CHIPS mimic, we designed and synthesized a substantially shorter 50-residue adapted peptide, designated CHOPS. This peptide included all residues important for receptor binding as based on the recent structure of CHIPS in complex with the C5aR N terminus. Using isothermal titration calorimetry we demonstrate that CHOPS has micromolar affinity for a model peptide comprising residues 7–28 of the C5aR N terminus including two O-sulfated tyrosine residues at positions 11 and 14. CD and NMR spectroscopy showed that CHOPS is unstructured free in solution. Upon addition of the doubly sulfated model peptide, however, the NMR and CD spectra reveal the formation of structural elements in CHOPS reminiscent of native CHIPS.Electronic supplementary materialThe online version of this article (doi:10.1007/s00726-010-0711-3) contains supplementary material, which is available to authorized users.