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J. Biol. Chem., Vol. 283, Issue 12, 7763-7775, March 21, 2008

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Structure of the Complement Factor 5a Receptor-Ligand Complex Studied by Disulfide Trapping and Molecular Modeling^*

Ian S. Hagemann, Daniel L. Miller, Jeffery M. Klco, Gregory V. Nikiforovich^¶, and Thomas J. Baranski¹

From the Departments of Medicine, Molecular Biology and Pharmacology, and ^¶Biochemistry and Molecular Biophysics, Washington University School of Medicine, St. Louis, Missouri 63110

Complement factor 5a (C5a) is an anaphylatoxin that acts by binding to a G protein-coupled receptor, the C5aR. The relative orientation of this ligand-receptor pair is investigated here using the novel technique of disulfide trapping by random mutagenesis (DTRM) and molecular modeling. In the DTRM technique, an unpaired cysteine is introduced in the ligand, and a library of randomly mutagenized receptors is screened to identify mutants that introduce a cysteine at a position in the receptor that allows functional interactions with the ligand. By repeating this analysis at six positions of C5a, we identify six unique sets of intermolecular interactions for the C5a-C5aR complex, which are then compared with an independently developed computational three-dimensional model of the complex. This analysis reveals that the interface of the receptor N terminus with the cysteine-containing ligand molecules is selected from a variety of possible receptor conformations that exist in dynamic equilibrium. In contrast, DTRM identifies a single position in the second extracellular loop of the receptor that interacts specifically with a cysteine probe placed in the C-terminal tail of the C5a ligand.

Received for publication, November 19, 2007 , and in revised form, January 8, 2008.

^* This work was supported by National Institutes of Health Grants GM71634 (to T. J. B. and G. V. N.), GM63720 (to T. J. B.), and GM22086 (to G. V. N.) and an American Heart Association predoctoral fellowship (to I. S. 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.

The on-line version of this article (available at http://www.jbc.org) contains supplemental "Experimental Procedures" and Tables S1 and S2.

¹ To whom correspondence should be addressed: 660 S. Euclid Ave., Campus Box 8127, St. Louis, MO 63110. E-mail: baranski{at}wustl.edu.

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