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.In recent years, Staphylococcus aureus has become a major health threat to both humans and domestic animals. It is found as a commensal bacterium in ∼30% of the human population, but when it becomes infectious it can cause a wide diversity of diseases, ranging from mild skin infections to life-threatening invasive conditions such as pneumonia and sepsis (1). Increased antibiotic resistance and a high amount of virulence factors secreted by S. aureus contribute to its emergence as a pathogen. Among these secreted virulence factors are the staphylococcal superantigen-like proteins (SSLs), a family of 14 proteins located on two genomic clusters (2–4). Recently, we and others identified SSL3 as a potent inhibitor of Toll-like receptor 2 (TLR2) (5, 6), an innate immunity receptor that is a dominant factor in immune recognition of S. aureus (7–10).TLR2 belongs to a family of 10 homologous innate immunity receptors that are activated by pathogen-associated molecular patterns (PAMPs) (11). TLR2 binds bacterial lipopeptides and lipoproteins. Subsequent formation of heterodimers with TLR1 or TLR6 leads to MyD88-dependent activation of the NF-κB pathway (12). TLR2 has dual ligand specificity that is determined by its dimerization partner; stimulation by diacyl lipopeptides from Gram-positive bacteria, including S. aureus, induces the formation of heterodimers with TLR6 (13), whereas triacyl lipopeptides from Gram-negative bacteria initiate formation of TLR2–TLR1 dimers (14). The structural basis for lipopeptide specificity was revealed by crystal structures of TLR2–TLR1 and TLR2–TLR6 complexes with their respective lipopeptide analogs Pam₃CSK₄ and Pam₂CSK₄: TLR2 binds two lipid tails in a large hydrophobic pocket, whereas the third lipid tail of triacyl lipopeptides is accommodated by a smaller pocket present in TLR1, but not in TLR6 (15, 16).The family of SSL proteins, including SSL3, share structural similarities to superantigens, but lack superantigenic activity. Interestingly, the functions that have been discovered for SSLs so far have all been linked to immune evasion. SSL5 inhibits neutrophil extravasation (17, 18) and phagocyte function (19, 20), SSL7 binds IgA and inhibits complement (21), and SSL10 inhibits IgG1-mediated phagocytosis (22, 23), blood coagulation (24), and the chemokine receptor CXCR4 (25). In addition to SSL3, also weak TLR2 inhibitory activity was observed for SSL4 (5), but it remains unknown whether that is its dominant function. This variety of immunomodulatory molecules and functions reflects the importance of the different components of our innate immune system in the defense against S. aureus (26).In this study we determined the crystal structures of SSL3 and the SSL3–TLR2 complex. In combination with mutagenesis and binding studies, our data provide a novel working mechanism of a functional TLR2 antagonist.