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J. Biol. Chem., Vol. 283, Issue 41, 27916-27926, October 10, 2008

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Paclitaxel Binding to Human and Murine MD-2^*

Shanta M. Zimmer¹, Jin Liu, Jaime L. Clayton, David S. Stephens, and James P. Snyder²

From the Department of Chemistry, Emory University, Atlanta, Georgia 30322 and the Division of Infectious Diseases, Emory University School of Medicine, Atlanta, Georgia 30322

Paclitaxel (PTX) is an important cancer chemotherapeutic agent that binds to β-tubulin and prevents mitosis through microtubule overstabilization. Recent evidence also implicates PTX in the induction of apoptosis of cancer cells via the TLR4 innate immune pathway. The TLR4 accessory protein, MD-2, is an essential component for the species-specific proinflammatory activity of PTX on murine cells. However, whether PTX binds to human MD-2 and how MD-2 and TLR4 interact with PTX are not well defined. Recombinant human MD-2 (rhMD-2) was produced in a Pichia pastoris expression system, and the interaction between rhMD-2 and PTX was assessed by an enzyme-linked immunosorbent assay to show that PTX binds rhMD-2. Formation of the latter complex was found to be dose-dependent and inhibited by anti-MD-2 antibody but not by an isotype control antibody. As measured by human tumor necrosis factor production, human THP-1 monocytes expressing TLR4 and MD-2 were poorly responsive to the addition of PTX, but murine macrophages expressing TLR4 and MD-2 responded in a dose-dependent manner. Human embryonic kidney (HEK293) cells transfected with both human TLR4 and human MD-2 or human MD-2 and murine TLR4 were also poorly responsive to PTX (10 µM). However, HEK293 cells transfected with murine MD-2 and human TLR4 or murine MD-2 and murine TLR4 were highly responsive to PTX (10 µM), indicating that the murine MD-2/PTX interaction is required for TLR4 activation. To further define the structural differences for MD-2/TLR4 activation, crystal structures of both murine and human MD-2 were subjected to PTX docking by computational methods. These models indicate that PTX binds in the pocket of both human and mouse MD-2 structures. The species-specific difference between human and murine MD-2 activation of TLR4 by PTX can be explained by alterations of surface charge distribution (i.e. electrostatic potential), binding pocket size, and the locus of PTX binding within the MD-2 pocket, which results in reorganization of the 123-130 amino acid loop. In particular, Phe¹²⁶ appears to operate as a bridge for TLR4·MD-2 dimerization in the mouse but not the human protein.

Received for publication, April 14, 2008 , and in revised form, June 23, 2008.

^* This work was supported, in whole or in part, by National Institutes of Health Grants K08 AI060966-03 (to S. M. Z.), R01 AI033517-10 (to D. S. S.), and R01 CA-69571 (to J. P. S.). 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 To whom correspondence may be addressed: Veterans Affairs Medical Center, Research Service 151, 1670 Clairmont Rd., Decatur, GA 30033, Tel.: 404-321-6111 (ext. 7570); Fax: 404-329-2210.

² To whom correspondence may be addressed: Dept. of Chemistry, Emory University, 1515 Dickey Dr., Atlanta, GA 30322. Tel.: 404-727-2415; Fax: 404-727-6586.

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