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Elucidation of the MD-2/TLR4 Interface Required for Signaling by Lipid IVa¹

Catherine Walsh^2,*, Monique Gangloff^2,, Tom Monie, Tomoko Smyth^*, Bin Wei^*, Trevelyan J. McKinley^*, Duncan Maskell^*, Nicholas Gay and Clare Bryant^3,*

^* Department of Veterinary Medicine and Department of Biochemistry, University of Cambridge, Cambridge, United Kingdom

LPS signals through a membrane bound-complex of the lipid binding protein MD-2 and the receptor TLR4. In this study we identify discrete regions in both MD-2 and TLR4 that are required for signaling by lipid IVa, an LPS derivative that is an agonist in horse but an antagonist in humans. We show that changes in the electrostatic surface potential of both MD-2 and TLR4 are required in order that lipid IVa can induce signaling. In MD-2, replacing horse residues 57–66 and 82–89 with the equivalent human residues confers a level of constitutive activity on horse MD-2, suggesting that conformational switching in this protein is likely to be important in ligand-induced activation of MD-2/TLR4. We identify leucine-rich repeat 14 in the C terminus of TLR4 as essential for lipid IVa activation of MD-2/TLR4. Remarkably, we identify a single residue in the glycan-free flank of the horse TLR4 solenoid that confers the ability to signal in response to lipid IVa. These results suggest a mechanism of signaling that involves crosslinking mediated by both MD-2-receptor and receptor-receptor contacts in a model that shows striking similarities to the recently published structure (Cell 130: 1071–1082) of the ligand-bound TLR1/2 ectodomain heterodimer.

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 C.W. was supported by a Horserace Betting Levy Board Scholarship and N.G. was supported by a Medical Research Council program grant.

² C.W. and M.G. contributed equally to this work.

³ Address correspondence and reprint requests to Dr. Clare Bryant, Department of Veterinary Medicine, University of Cambridge, Madingley Road, Cambridge, CB3 OES, U.K. E-mail address: ceb27{at}cam.ac.uk

⁴ Abbreviations used in this paper: LRR, leucine-rich repeat; EH, chimera in which a human region is engineered into an equine framework; HE, chimera in which an equine region is engineered into a human framework.

⁵ The online version of this article contains supplemental material.