title: Mapping of a microbial protein domain involved in binding and activation of the TLR2/TLR1 heterodimer.
abstract: The pentameric B subunit of type IIb Escherichia coli enterotoxin (LT-IIb-B(5)), 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-B(5) interaction with TLR2/1 to define the structure-function relationship of LT-IIb-B(5) 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 Pam(3)CysSerLys(4) (Pam(3)CSK(4)) lipopeptide. We first identified four critical residues in the upper region of the LT-IIb-B(5) 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-Pam(3)CSK(4) complex, resulted in diminished activation by both Pam(3)CSK(4) and LT-IIb-B(5). Docking analysis of the LT-IIb-B(5) interaction with this apparently predominant activation conformation of TLR2/1 revealed that LT-IIb-B(5) might primarily contact the convex surface of the TLR2 central domain. Although the TLR1/LT-IIb-B(5) 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-B(5). Moreover, the putative LT-IIb-B(5) binding site overlaps partially with that of Pam(3)CSK(4); consistent with this, Pam(3)CSK(4) suppressed TLR2 binding of LT-IIb-B(5), albeit not as potently as self-competitive inhibition. We identified the upper pore region of LT-IIb-B(5) as a TLR2/1 interactive domain, which contacts the heterodimeric receptor at a site that is distinct from, although it overlaps with, that of Pam(3)CSK(4).
authors.affiliation: Center for Oral Health and Systemic Disease, University of Louisville Health Sciences Center, Louisville, KY 40292, USA.
authors.forename: Shuang
authors.initials: S
authors.lastname: Liang
authors.forename: Kavita B
authors.initials: KB
authors.lastname: Hosur
authors.forename: Shanyun
authors.initials: S
authors.lastname: Lu
authors.forename: Hesham F
authors.initials: HF
authors.lastname: Nawar
authors.forename: Benjamin R
authors.initials: BR
authors.lastname: Weber
authors.forename: Richard I
authors.initials: RI
authors.lastname: Tapping
authors.forename: Terry D
authors.initials: TD
authors.lastname: Connell
authors.forename: George
authors.initials: G
authors.lastname: Hajishengallis
mesh_terms: D000818:Animals; D001427:Bacterial Toxins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D045744:Cell Line, Tumor; D002478:Cells, Cultured; D018360:Crystallography, X-Ray; D019281:Dimerization; D054307:Enterotoxigenic Escherichia coli / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D004768:Enterotoxins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D029968:Escherichia coli Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D055666:Lipopeptides / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D051379:Mice; D008807:Mice, Inbred BALB C; D010449:Peptide Mapping / Q000379:methods; D017354:Point Mutation; D011485:Protein Binding / Q000235:genetics / Q000276:immunology; D021122:Protein Subunits / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D051194:Toll-Like Receptor 1 / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000378:metabolism*; D051195:Toll-Like Receptor 2 / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000378:metabolism*
chemical_list: D001427:Bacterial Toxins; D004768:Enterotoxins; D029968:Escherichia coli Proteins; D055666:Lipopeptides; D021122:Protein Subunits; C495342:TLR2 protein, human; D051194:Toll-Like Receptor 1; D051195:Toll-Like Receptor 2; C027538:heat-labile enterotoxin, E coli