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J. Biol. Chem., Vol. 282, Issue 13, 10005-10017, March 30, 2007

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Critical Role for Polar Residues in Coupling Leukotriene B₄ Binding to Signal Transduction in BLT1^*

Sudeep Basu¹, Venkatakrishna R. Jala, Steven Mathis, Soujanya T. Rajagopal, Annalisa Del Prete, Paramahamsa Maturu, John O. Trent^¶2, and Bodduluri Haribabu³

From the Tumor Immunobiology Program, James Graham Brown Cancer Center and Departments of Microbiology and Immunology and of ^¶Medicine, University of Louisville Health Sciences, Louisville, Kentucky 40202

Leukotriene B₄ (LTB₄) mediates a variety of inflammatory diseases such as asthma, arthritis, atherosclerosis, and cancer through activation of the G-protein-coupled receptor, BLT1. Using in silico molecular dynamics simulations combined with site-directed mutagenesis we characterized the ligand binding site and activation mechanism for BLT1. Mutation of residues predicted as potential ligand contact points in transmembrane domains (TMs) III (H94A and Y102A), V (E185A), and VI (N241A) resulted in reduced binding affinity. Analysis of arginines in extracellular loop 2 revealed that mutating arginine 156 but not arginine 171 or 178 to alanine resulted in complete loss of LTB₄ binding to BLT1. Structural models for the ligand-free and ligand-bound states of BLT1 revealed an activation core formed around Asp-64, displaying multiple dynamic interactions with Asn-36, Ser-100, and Asn-281 and a triad of serines, Ser-276, Ser-277, and Ser-278. Mutagenesis of many of these residues in BLT1 resulted in loss of signaling capacity while retaining normal LTB₄ binding function. Thus, polar residues within TMs III, V, and VI and extracellular loop 2 are critical for ligand binding, whereas polar residues in TMs II, III, and VII play a central role in transducing the ligand-induced conformational change to activation. The delineation of a validated binding site and activation mechanism should facilitate structure-based design of inhibitors targeting BLT1.

Received for publication, October 10, 2006 , and in revised form, January 11, 2007.

^* This work was supported in part by National Institutes of Health Grant AI52381 (to B. H.). 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 Supported by an American Heart Association Pre-doctoral award.

² To whom correspondence may be addressed: The James Graham Brown Cancer Center, University of Louisville, 529 South Jackson St., Louisville, KY 40202. Tel.: 502-852-2194; Fax: 502-852-4311; E-mail: john.trent{at}louisville.edu.

³ To whom correspondence may be addressed: The James Graham Brown Cancer Center, University of Louisville, 580 South Preston St., Louisville, KY 40202. Tel.: 502-852-7503; Fax: 502-852-2123; E-mail: H0bodd01{at}gwise.louisville.edu.

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