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J. Biol. Chem., Vol. 282, Issue 45, 33118-33131, November 9, 2007

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Structural and Thermodynamic Bases for the Design of Pure Prolactin Receptor Antagonists

X-RAY STRUCTURE OF Del1-9-G129R-hPRL^*

Jean-Baptiste Jomain¹², Estelle Tallet¹, Isabelle Broutin^¶, Sylviane Hoos^||, Jan van Agthoven^¶1, Arnaud Ducruix^¶, Paul A. Kelly, Birthe B. Kragelund^**, Patrick England^||, and Vincent Goffin³

From the Inserm, U845, Centre de Recherche "Croissance et Signalisation," Equipe "PRL, GH et Tumeurs," Paris F-75015, France, Université Paris Descartes, FacultédeMédecine, Paris, F-75015 France, ^¶Laboratoire de Cristallographie et RMN Biologiques CNRS, UMR 8015, Université Paris Descartes, 75006, Paris, France, ^||Institut Pasteur, Plateforme de Biophysique des Macromolécules et de Leurs Interactions, Département de Biologie Structurale et Chimie, 75015, Paris, France, and ^**Structural Biology and NMR Laboratory, Department of Molecular Biology, University of Copenhagen, DK-2200 Copenhagen, Denmark

Competitive antagonists of the human prolactin (hPRL) receptor are a novel class of molecules of potential therapeutic interest in the context of cancer. We recently developed the pure antagonist Del1-9-G129R-hPRL by deleting the nine N-terminal residues of G129R-hPRL, a first generation partial antagonist. We determined the crystallographic structure of Del1-9-G129R-hPRL, which revealed no major change compared with wild type hPRL, indicating that its pure antagonistic properties are intrinsically due to the mutations. To decipher the molecular bases of pure antagonism, we compared the biological, physicochemical, and structural properties of numerous hPRL variants harboring N-terminal or Gly¹²⁹ mutations, alone or combined. The pure versus partial antagonistic properties of the multiple hPRL variants could not be correlated to differences in their affinities toward the hPRL receptor, especially at site 2 as determined by surface plasmon resonance. On the contrary, residual agonism of the hPRL variants was found to be inversely correlated to their thermodynamic stability, which was altered by all the Gly¹²⁹ mutations but not by those involving the N terminus. We therefore propose that residual agonism can be abolished either by further disrupting hormone site 2-receptor contacts by N-terminal deletion, as in Del1-9-G129R-hPRL, or by stabilizing hPRL and constraining its intrinsic flexibility, as in G129V-hPRL.

Received for publication, May 29, 2007 , and in revised form, August 14, 2007.

The atomic coordinates and structure factors (code 2Q98) have been deposited in the Protein Data Bank, Research Collaboratory for Structural Bioinformatics, Rutgers University, New Brunswick, NJ (http://www.rcsb.org/).

^* This work was supported in part by INSERM, University Paris Descartes, the Comité de Paris de la Ligue Nationale Contre le Cancer Grant R05/75-15, and the Danish Natural Research Council Grant 21040604. 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 Recipient of support from the Ministère de l'Education Nationale, de la Recherche et de la Technologie.

² Recipient of fellowship support from the Association pour la Recherche sur le Cancer.

³ To whom correspondence should be addressed: INSERM U845, Centre de Recherche Croissance et Signalisation, Facultéde Médecine Necker, 156 Rue de Vaugirard, 75730, Paris Cedex 15, France. Tel.: 331-40-61-56-16; Fax: 331-43-06-04-43; E-mail: goffin{at}necker.fr.

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