Mapping of a microbial protein domain involved in binding and activation of the TLR2/TLR1 heterodimer

The pentameric B subunit of type IIb Escherichia coli enterotoxin (LT-IIb-B₅), a doughnut-shaped oligomeric protein from enterotoxigenic E. coli, activates the TLR2/TLR1 heterodimer (TLR2/1). We investigated the molecular basis of the LTIIb-B₅ interaction with TLR2/1 to define the structure-function relationship of LT-IIb-B₅ and, moreover, to gain an insight into how TLR2/1 recognizes large, nonacylated protein ligands that cannot fit within its lipid-binding pockets, as previously shown for the Pam₃CysSerLys₄ (Pam₃CSK ₄) lipopeptide. We first identified four critical residues in the upper region of the LT-IIb-B₅ pore. Corresponding point mutants (M69E, A70D, L73E, S74D) were defective in binding TLR2 or TLR1 and could not activate APCs, despite retaining full ganglioside-binding capacity. Point mutations in the TLR2/1 dimer interface, as determined in the crystallographic structure of the TLR2/1-Pam₃CSK₄ complex, resulted in diminished activation by both Pam₃CSK₄ and LT-IIb-B ₅. Docking analysis of the LT-IIb-B₅ interaction with this apparently predominant activation conformation of TLR2/1 revealed that LT-IIb-B₅ might primarily contact the convex surface of the TLR2 central domain. Although the TLR1/LT-IIb-B₅ interface is relatively smaller, the leucine-rich repeat motifs 9 -12 in the central domain of TLR1 were found to be critical for cooperative TLR2-induced cell activation by LT-IIb-B₅. Moreover, the putative LT-IIb-B₅ binding site overlaps partially with that of Pam₃CSK₄; consistent with this, Pam₃CSK₄ suppressed TLR2 binding of LTIIb-B ₅, albeit not as potently as self-competitive inhibition. We identified the upper pore region of LT-IIb-B₅ as a TLR2/1 interactive domain, which contacts the heterodimeric receptor at a site that is distinct from, although it overlaps with, that of Pam₃CSK₄.