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J. Biol. Chem., Vol. 281, Issue 31, 22378-22385, August 4, 2006

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Comprehensive and Quantitative Mapping of Energy Landscapes for Protein-Protein Interactions by Rapid Combinatorial Scanning^*

Gábor Pál¹, Jean-Louis K. Kouadio, Dean R. Artis, Anthony A. Kossiakoff², and Sachdev S. Sidhu³

From the Department of Protein Engineering, Genentech Inc., South San Francisco, California 94080 and the Department of Biochemistry and Molecular Biology and Institute for Biophysical Dynamics, Cummings Life Sciences Center, University of Chicago, Chicago, Illinois 60637

A novel, quantitative saturation (QS) scanning strategy was developed to obtain a comprehensive data base of the structural and functional effects of all possible mutations across a large protein-protein interface. The QS scan approach was applied to the high affinity site of human growth hormone (hGH) for binding to its receptor (hGHR). Although the published structure-function data base describing this system is probably the most extensive for any large protein-protein interface, it is nonetheless too sparse to accurately describe the nature of the energetics governing the interaction. Our comprehensive data base affords a complete view of the binding site and provides important new insights into the general principles underlying protein-protein interactions. The hGH binding interface is highly adaptable to mutations, but the nature of the tolerated mutations challenges generally accepted views about the evolutionary and biophysical pressures governing protein-protein interactions. Many substitutions that would be considered chemically conservative are not tolerated, while conversely, many non-conservative substitutions can be accommodated. Furthermore, conservation across species is a poor predictor of the chemical character of tolerated substitutions across the interface. Numerous deviations from generally accepted expectations indicate that mutational tolerance is highly context dependent and, furthermore, cannot be predicted by our current knowledge base. The type of data produced by the comprehensive QS scan can fill the gaps in the structure-function matrix. The compilation of analogous data bases from studies of other protein-protein interactions should greatly aid the development of computational methods for explaining and designing molecular recognition.

Received for publication, April 20, 2006 , and in revised form, June 6, 2006.

^* 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 Tables S1 and S2.

This article was selected as a Paper of the Week.

¹ Present address: Dept. of Biochemistry, Eötvös University, 1/C Pázmány P. Street, Budapest, Hungary.

² To whom correspondence may be addressed: Dept. of Biochemistry and Molecular Biology and Inst. for Biophysical Dynamics, Cummings Life Sciences Center, University of Chicago, 920 East 58th St., Chicago, IL 60637. Tel.: 773-702-9257; Fax: 773-702-0439; E-mail: koss{at}cummings.uchicago.

³ To whom correspondence may be addressed: Dept. of Protein Engineering, Genentech Inc., 1 DNA Way, South San Francisco, CA 94080. Tel.: 650-225-1056; Fax: 650-225-3734; E-mail: sidhu{at}gene.com.

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