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Asp218 participates with Asp213 to bind a Ca2+ atom into the S1 subsite of aminopeptidase A: a key element for substrate specificity.

Cédric Claperon 1 Raphael Rozenfeld 1 Xavier Iturrioz 1 Nicolas Inguimbert 2 Mayumi Okada 1 Bernard P. Roques 2 Bernard Maigret 3 Catherine Llorens-Cortes 1 
1 Neuropeptides centraux et régulations hydrique et cardiovasculaire
UPMC - Université Pierre et Marie Curie - Paris 6, CIRB - Centre interdisciplinaire de recherche en biologie, INSERM - Institut National de la Santé et de la Recherche Médicale : U691
3 ORPAILLEUR - Knowledge representation, reasonning
INRIA Lorraine, LORIA - Laboratoire Lorrain de Recherche en Informatique et ses Applications
Abstract : APA (aminopeptidase A; EC is a membrane-bound zinc metallopeptidase, also activated by Ca(2+), involved in the formation of brain angiotensin III, which exerts a tonic stimulatory action on the central control of blood pressure in hypertensive animals. In the present study, in the three-dimensional model of the ectodomain of mouse APA, we docked the specific APA inhibitor glutamate phosphonate, in the presence of Ca(2+). The model showed the presence of one Ca(2+) atom in an hydrophilic pocket corresponding to the S1 subsite in which the lateral chain of the inhibitor is pointing. In this pocket, the Ca(2+) atom was hexaco-ordinated with the acidic side chains of Asp(213) and Asp(218), the carbonyl group of Glu(215) and three water molecules, one of them being engaged in a hydrogen bond with the negatively charged carboxylate side chain of the inhibitor. Mutagenic replacement of Asp(213) and Asp(218) with a conservative residue maintained the ability of mutated APAs to be activated by Ca(2+). However, the replacement by a non-conservative residue abolished this property, demonstrating the crucial role of these residues in Ca(2+) binding. We also showed the involvement of these residues in the strict specificity of APA in the presence of Ca(2+) for N-terminal acidic residues from substrates or inhibitors, since mutagenic replacement of Asp(213) and Asp(218) induced a decrease of the inhibitory potencies of inhibitors homologous with acidic residues. Finally, this led to the rational design of a new potent APA inhibitor, NI926 (K(i)=70 nM), which allowed us to precisely localize Asp(213) at the entrance and Asp(218) at the bottom of the S1 subsite. Taken together, these data provide new insight into the organization and functional role of the APA S1 subsite and will allow the design of pharmacophore of the inhibitor, helpful for the development of a new generation of APA inhibitors as central-acting antihypertensive agents.
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Submitted on : Thursday, October 8, 2009 - 11:14:29 AM
Last modification on : Thursday, March 17, 2022 - 10:08:35 AM

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Cédric Claperon, Raphael Rozenfeld, Xavier Iturrioz, Nicolas Inguimbert, Mayumi Okada, et al.. Asp218 participates with Asp213 to bind a Ca2+ atom into the S1 subsite of aminopeptidase A: a key element for substrate specificity.. Biochemical Journal, Portland Press, 2008, 416 (1), pp.37-46. ⟨10.1042/BJ20080471⟩. ⟨inserm-00422659⟩



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