Asp-218 participates with asp-213 to bind a ca2+ atom into the S1 subsite of aminopeptidase A: a key element for substrate specificity - Inserm - Institut national de la santé et de la recherche médicale Access content directly
Journal Articles Biochemical Journal Year : 2008

Asp-218 participates with asp-213 to bind a ca2+ atom into the S1 subsite of aminopeptidase A: a key element for substrate specificity

Cédric Claperon
  • Function : Author
Raphael Rozenfeld
  • Function : Author
Xavier Iturrioz
  • Function : Author
Nicolas Inguimbert
Mayumi Okada
  • Function : Author
Bernard P. Roques
  • Function : Author
Bernard Maigret
  • Function : Author

Abstract

Aminopeptidase A (EC 3.4.11.7, APA) 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 three-dimentional 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 into an hydrophilic pocket corresponding to the S1 subsite in which the lateral chain of inhibitor is pointing. In this pocket, the Ca2+ atom was hexacoordinated 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 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 Asp-213 and Asp-218 induced a decrease of the inhibitory potencies of inhibitors homologous to acidic residues. Finally, this led to rationally design a new potent APA inhibitor, NI926 (Ki = 70 nM), which allowed to precisely localize Asp-213 at the entrance and Asp-218 at the bottom of the S1 subsite. Altogether, these data provide new insight into the organization and functional role of the APA S1 subsite and will allow to design a pharmacophore of inhibitor, helpful for the development of a new generation of APA inhibitors as central-acting antihypertensive agents.

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Dates and versions

hal-00478991 , version 1 (30-04-2010)

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Cédric Claperon, Raphael Rozenfeld, Xavier Iturrioz, Nicolas Inguimbert, Mayumi Okada, et al.. Asp-218 participates with asp-213 to bind a ca2+ atom into the S1 subsite of aminopeptidase A: a key element for substrate specificity. Biochemical Journal, 2008, 416 (1), pp.37-46. ⟨10.1042/BJ20080471⟩. ⟨hal-00478991⟩
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