Mapping of a Microbial Protein Domain Involved in Binding and Activation of the TLR2/TLR1 Heterodimer1

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 LT-IIb-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 (Pam3CSK4) 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-B5. 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 LT-IIb-B5, 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.