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

Shuang Liang^*, Kavita B. Hosur^*, Shanyun Lu, Hesham F. Nawar, Benjamin R. Weber, Richard I. Tapping, Terry D. Connell and George Hajishengallis^2,*,¶

^* Center for Oral Health and Systemic Disease, University of Louisville Health Sciences Center, Louisville, KY 40292; Center for Biophysical Science and Engineering, University of Alabama, Birmingham, AL 35294, Department of Microbiology and Immunology, Witebsky Center for Microbial Pathogenesis and Immunology, State University of New York at Buffalo, Buffalo, NY 14214; Department of Microbiology and The College of Medicine, University of Illinois, Urbana, IL 61801; and ^¶ Department of Microbiology and Immunology, University of Louisville Health Sciences Center, Louisville, KY 40292

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.

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

¹ This work was supported by National Institutes of Health R01 Grants AI052344 and AI056148 (to R.I.T.), DE013833 (to T.D.C.), and DE015254 and DE017138 (to G.H.).

² Address correspondence and reprint requests to Dr. George Hajishengallis, University of Louisville Health Sciences Center, 501 South Preston Street, Room 206, Louisville, KY 40292. E-mail address: g0haji01{at}louisville.edu

³ Abbreviations used in this paper: LT-IIb, type IIb enterotoxin of E. coli; LT-IIb-B₅, pentameric B subunit of LT-IIb; LRR, leucine-rich repeat; Pam₃CSK₄, Pam₃CysSerLys₄; BMDC, bone marrow-derived dendritic cell.

⁴ The online version of this article contains supplemental material.