APA (aminopeptidase A; EC 3.4.11.7) is a membrane-bound zinc metallopeptidase, also activated by Ca2+, 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 Ca2+. The model showed the presence of one Ca2+ atom in an hydrophilic pocket corresponding to the S1 subsite in which the lateral chain of the inhibitor is pointing. In this pocket, the Ca2+ atom was hexaco-ordinated with the acidic side chains of Asp213 and Asp218, the carbonyl group of Glu215 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 Asp213 and Asp218 with a conservative residue maintained the ability of mutated APAs to be activated by Ca2+. However, the replacement by a non-conservative residue abolished this property, demonstrating the crucial role of these residues in Ca2+ binding. We also showed the involvement of these residues in the strict specificity of APA in the presence of Ca2+ for N-terminal acidic residues from substrates or inhibitors, since mutagenic replacement of Asp213 and Asp218 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 (Ki=70 nM), which allowed us to precisely localize Asp213 at the entrance and Asp218 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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Research Article|
October 28 2008
Asp218 participates with Asp213 to bind a Ca2+ atom into the S1 subsite of aminopeptidase A: a key element for substrate specificity
Cédric Claperon;
Cédric Claperon
*INSERM, U691, Paris, FR-75005 France
†Collège de France, Paris, FR-75005 France
‡Université Pierre et Marie Curie, Paris, FR-75005 France
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Raphael Rozenfeld;
Raphael Rozenfeld
*INSERM, U691, Paris, FR-75005 France
†Collège de France, Paris, FR-75005 France
‡Université Pierre et Marie Curie, Paris, FR-75005 France
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Xavier Iturrioz;
Xavier Iturrioz
*INSERM, U691, Paris, FR-75005 France
†Collège de France, Paris, FR-75005 France
‡Université Pierre et Marie Curie, Paris, FR-75005 France
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Nicolas Inguimbert;
Nicolas Inguimbert
§INSERM, U640, Paris, FR-75005 France
∥Université René Descartes, Paris, FR-75005 France
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Mayumi Okada;
Mayumi Okada
*INSERM, U691, Paris, FR-75005 France
†Collège de France, Paris, FR-75005 France
‡Université Pierre et Marie Curie, Paris, FR-75005 France
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Bernard Roques;
Bernard Roques
§INSERM, U640, Paris, FR-75005 France
∥Université René Descartes, Paris, FR-75005 France
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Bernard Maigret;
Bernard Maigret
¶CNRS, UMR 7503, Vandoeuvre Les Nancy, FR-54506, France; Laboratoire Lorrain de Recherche en Informatique et ses applications (LORIA)
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Catherine Llorens-Cortes
Catherine Llorens-Cortes
1
*INSERM, U691, Paris, FR-75005 France
†Collège de France, Paris, FR-75005 France
‡Université Pierre et Marie Curie, Paris, FR-75005 France
1To whom correspondence should be addressed (email c.llorens-cortes@college-de-france.fr).
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Publisher: Portland Press Ltd
Received:
February 29 2008
Revision Received:
June 30 2008
Accepted:
July 04 2008
Accepted Manuscript online:
July 04 2008
Online ISSN: 1470-8728
Print ISSN: 0264-6021
© The Authors Journal compilation © 2008 Biochemical Society
2008
Biochem J (2008) 416 (1): 37–46.
Article history
Received:
February 29 2008
Revision Received:
June 30 2008
Accepted:
July 04 2008
Accepted Manuscript online:
July 04 2008
Citation
Cédric Claperon, Raphael Rozenfeld, Xavier Iturrioz, Nicolas Inguimbert, Mayumi Okada, Bernard Roques, Bernard Maigret, Catherine Llorens-Cortes; Asp218 participates with Asp213 to bind a Ca2+ atom into the S1 subsite of aminopeptidase A: a key element for substrate specificity. Biochem J 15 November 2008; 416 (1): 37–46. doi: https://doi.org/10.1042/BJ20080471
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