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J. Biol. Chem., Vol. 280, Issue 27, 25524-25532, July 8, 2005

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Shotgun Alanine Scanning Shows That Growth Hormone Can Bind Productively to Its Receptor through a Drastically Minimized Interface^*

Jean-Louis K. Kouadio, James R. Horn¶, Gabor Pal||, and Anthony A. Kossiakoff**

From the Department of Biochemistry and Molecular Biology and the ^**Institute for Biophysical Dynamics, the University of Chicago, Chicago, Illinois 60637

The high affinity binding site (Site1) of the human growth hormone (hGH) binds to its cognate receptor (hGHR) via a concave surface patch containing about 35 residues. Using 167 sequences from a shotgun alanine scanning analysis of Site1, we have determined that over half of these residues can be simultaneously changed to an alanine or a non-isosteric amino acid while still retaining a high affinity interaction. Among these hGH variants the distribution of the mutation is highly variable throughout the interface, although helix 4 is more conserved than the other binding elements. Kinetic and thermodynamic analyses were performed on 11 representative hGH Site1 variants that contained 14–20 mutations. Generally, the tightest binding variants showed similar associated rate constants (k_on) as the wild-type (wt) hormone, indicating that their binding proceeds through a similar transition state intermediate. However, calorimetric analyses indicate very different thermodynamic partitioning: wt-hGH binding exhibits favorable enthalpy and entropy contributions, whereas the variants display highly favorable enthalpy and highly unfavorable entropy contributions. The heat capacities (C_p) on binding measured for wt-hGH and its variants are significantly larger than normally seen for typical protein-protein interactions, suggesting large conformational or solvation effects. The multiple Site1 mutations are shown to indirectly affect binding of the second receptor at Site2 through an allosteric mechanism. We show that the stability of the ternary hormone-receptor complex reflects the affinity of the Site2 binding and is surprisingly exempt from changes in Site1 affinity, directly demonstrating that dissociation of the active signaling complex is a stepwise process.

Received for publication, February 25, 2005 , and in revised form, April 26, 2005.

^* This work was supported in part by Grant DK-61602 from the National Institutes for Health (NIH) (to A. A. K.). 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.

Supported by Minority Research Supplement Grant 3-R01-DK61602-02S1 from the NIDDK, NIH.

^¶ Supported by postdoctoral fellowships from the Northwestern University Drug Discovery Program and the American Heart Association.

^|| Present address: Dept. of Biochemistry, Eotvos University, 1/C Pazmany P. Street, Budapest, Hungary.

To whom correspondence should be addressed: Dept. of Biochemistry and Molecular Biology and Institute for Biophysical Dynamics, University of Chicago, Cummings Life Science Center, 920 East 58th St., Chicago, IL 60637. Tel.: 773-702-9257; Fax: 773-834-2777; E-mail: koss{at}cummings.uchicago.edu.

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