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J. Biol. Chem., Vol. 282, Issue 42, 30658-30666, October 19, 2007

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Mutations Designed to Destabilize the Receptor-Bound Conformation Increase MICA-NKG2D Association Rate and Affinity^*

Candice S. E. Lengyel, Lindsey J. Willis, Patrick Mann, David Baker, Tanja Kortemme^¶, Roland K. Strong^||, and Benjamin J. McFarland¹

From the Department of Chemistry and Biochemistry, Seattle Pacific University, Seattle, Washington 98119, the Department of Biochemistry, University of Washington, Seattle, Washington 98195, the ^¶Department of Biopharmaceutical Sciences, University of California, San Francisco, California 94143, and the ^||Division of Basic Sciences, Fred Hutchinson Cancer Research Center, Seattle, Washington 98109

MICA is a major histocompatibility complex-like protein that undergoes a structural transition from disorder to order upon binding its immunoreceptor, NKG2D. We redesigned the disordered region of MICA with RosettaDesign to increase NKG2D binding. Mutations that stabilize this region were expected to increase association kinetics without changing dissociation kinetics, increase affinity of interaction, and reduce entropy loss upon binding. MICA mutants were stable in solution, and they were amenable to surface plasmon resonance evaluation of NKG2D binding kinetics and thermodynamics. Several MICA mutants bound NKG2D with enhanced affinity, kinetic changes were primarily observed during association, and thermodynamic changes in entropy were as expected. However, none of the 15 combinations of mutations predicted to stabilize the receptor-bound MICA conformation enhanced NKG2D affinity, whereas all 10 mutants predicted to be destabilized bound NKG2D with increased on-rates. Five of these had affinities enhanced by 0.9–1.8 kcal/mol over wild type by one to three non-contacting substitutions. Therefore, in this case, mutations designed to mildly destabilize a protein enhanced association and affinity.

Received for publication, June 1, 2007 , and in revised form, July 30, 2007.

^* 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 should be addressed: 3307 3rd Ave. W, Suite 205, Seattle, WA 98119-1997. Tel.: 206-281-2749; Fax: 206-281-2882; E-mail: bjm{at}spu.edu.

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