Structural insights into collagen binding by platelet receptor glycoprotein VI.

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 ß-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 peptides (CRPs). 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 ≥2 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.

Molecular basis determining species specificity for TLR2 inhibition by staphylococcal superantigen-like protein 3 (SSL3).

Staphylococcus aureus is a versatile opportunistic pathogen, causing disease in human and animal species. Its pathogenicity is linked to the ability of S. aureus to secrete immunomodulatory molecules. These evasion proteins bind to host receptors or their ligands, resulting in inhibitory effects through high affinity protein-protein interactions. Staphylococcal evasion molecules are often species-specific due to differences in host target proteins between species. We recently solved the crystal structure of murine TLR2 in complex with immunomodulatory molecule staphylococcal superantigen-like protein 3 (SSL3), which revealed the essential residues within SSL3 for TLR2 inhibition. In this study we aimed to investigate the molecular basis of the interaction on the TLR2 side. The SSL3 binding region on murine TLR2 was compared to that of other species through sequence alignment and homology modeling, which identified interspecies differences. To examine whether this resulted in altered SSL3 activity on the corresponding TLR2s, bovine, equine, human, and murine TLR2 were stably expressed in HEK293T cells and the ability of SSL3 to inhibit TLR2 was assessed. We found that SSL3 was unable to inhibit bovine TLR2. Subsequent loss and gain of function mutagenesis showed that the lack of inhibition is explained by the absence of two tyrosine residues in bovine TLR2 that play a prominent role in the SSL3-TLR2 interface. We found no evidence for the existence of allelic SSL3 variants that have adapted to the bovine host. Thus, within this paper we reveal the molecular determinants of the TLR2-SSL3 interaction which adds to our understanding of staphylococcal host specificity.

Betacoronavirus Adaptation to Humans Involved Progressive Loss of Hemagglutinin-Esterase Lectin Activity.

Human beta1-coronavirus (ß1CoV) OC43 emerged relatively recently through a single zoonotic introduction. Like related animal ß1CoVs, OC43 uses 9-O-acetylated sialic acid as receptor determinant. ß1CoV 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.

Coronavirus receptor switch explained from the stereochemistry of protein-carbohydrate interactions and a single mutation.

Hemagglutinin-esterases (HEs) are bimodular envelope proteins of orthomyxoviruses, toroviruses, and coronaviruses with a carbohydrate-binding "lectin" domain appended to a receptor-destroying 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 protein-carbohydrate 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.

Structural basis for inhibition of TLR2 by staphylococcal superantigen-like protein 3 (SSL3).

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 Pam2CSK4 effectively, yet allows SSL3 to bind to an already formed TLR2-Pam2CSK4 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.

CHIPS binds to the phosphorylated N-terminus of the C5a-receptor.

Replacement of the sulfate groups, present in vivo on the N-terminus of the C5a-receptor (C5aR), by phosphate groups is explored. Phosphorylated mimics of the C5a-receptor N-terminus are synthesized and their binding to Chemotaxis Inhibitory Protein of Staphylococcus aureus (CHIPS) is studied by ITC and NMR. The phosphorylated C5aR mimics showed comparable binding affinity and a similar binding mode towards CHIPS compared to their sulfated forms. The activities of the phosphorylated peptides in a biological assay, however, were significantly lower compared to their sulfated counterparts.