Structural basis of peptide recognition by the angiotensin-1 converting enzyme homologue AnCE from Drosophila melanogaster

Abstract

Human somatic angiotensin-1 converting enzyme (ACE) is a zinc-dependent exopeptidase, that catalyses the conversion of the decapeptide angiotensin I to the octapeptide angiotensin II, by removing a C-terminal dipeptide. It is the principal component of the renin-angiotensin-aldosterone system that regulates blood pressure. Hence it is an important therapeutic target for the treatment of hypertension and cardiovascular disorders. Here, we report the structures of an ACE homologue from Drosophila melanogaster (AnCE; a proven structural model for the more complex human ACE) co-crystallized with mammalian peptide substrates (bradykinin, Thr(6) -bradykinin, angiotensin I and a snake venom peptide inhibitor, bradykinin-potentiating peptide-b). The structures determined at 2-Å resolution illustrate that both angiotensin II (the cleaved product of angiotensin I by AnCE) and bradykinin-potentiating peptide-b bind in an analogous fashion at the active site of AnCE, but also exhibit significant differences. In addition, the binding of Arg-Pro-Pro, the cleavage product of bradykinin and Thr(6) - bradykinin, provides additional detail of the general peptide binding in AnCE. Thus the new structures of AnCE complexes presented here improves our understanding of the binding of peptides and the mechanism by which peptides inhibit this family of enzymes.

Database: The atomic coordinates and structure factors for AnCE-Ang II (code 4AA1), AnCE-BPPb (code 4AA2), AnCE-BK (code 4ASQ) and AnCE-Thr6-BK (code 4ASR) complexes have been deposited in the Protein Data Bank, Research Collaboratory for Structural Bioinformatics, Rutgers University, New Brunswick, NJ (http://www.rcsb.org/)

Structured digital abstract: • AnCE cleaves Ang I by enzymatic study (View interaction) • Bradykinin and AnCE bind by x-ray crystallography (View interaction) • BPP and AnCE bind by x-ray crystallography (View interaction) • AnCE cleaves Bradykinin by enzymatic study (View interaction) • Ang II and AnCE bind by x-ray crystallography (View interaction).

Fig. 1

Substrate-bound Drosophila melanogaster AnCE crystal structure. AnCE (cyan) in cartoon representation, with Ang II as red sticks, glycosylation carbohydrates as yellow sticks. The catalytic zinc ion is shown as an olive green sphere.

Fig. 2

Portions of the difference electron density map for the bound peptide in the active site of AnCE. Electron density map is contoured at 1σ level. The picture was created using a Fourier difference density map in which the peptide atoms were omitted (A) Ang II, (B) BK, (C) Thr⁶–BK and (D) BPPb in the crystal structure of their respective complex with AnCE.

Fig. 3

AnCE–peptide interactions. (A) Ang II-bound AnCE crystal structure. AnCE (cyan), with Ang II in red sticks. The catalytic zinc ion is shown as a green sphere. Bound water molecules as small spheres. (B) Schematic view of Ang II binding. Hydrophobic interactions, hydrogen bonds and distances cited (grey). (C) BK-bound AnCE crystal structure. AnCE (cyan), with BK in pink sticks. Citrate ion in grey. (D) Schematic view of BK binding. Hydrophobic interactions, hydrogen bonds and distances cited (grey). (E) BPPb-bound AnCE crystal structure. AnCE (cyan) with BPPb in orange sticks. (F) Schematic view of BPPb binding. Hydrophobic interactions, hydrogen bonds and distances cited (grey).

Fig. 4

Inhibition of Drosophila melanogaster AnCE by Ang II. AnCE was assayed using HHL as substrate in presence of various concentrations of Ang II as described in the Experimental procedures. (A) Data are expressed as a percentage of uninhibited activity and each data point is the mean of three replicates. (B) Lineweaver–Burk plot showing the competitive nature of the inhibition by Ang II of the hydrolysis of HHL by AnCE. (C) The effect of Ang II on the hydrolysis of Ang I (100 μm) by AnCE was determined by quantifying the amount of Ang I consumed in the reaction in the absence and presence of 100 μm Ang II. The results are expressed as % hydrolysis occurring in 30 min and are means ± SEM (n = 4).

Fig. 5

Comparison of AnCE–Ang II interactions. (A) Comparison of Ang II binding in AnCE and the C-domain human somatic ACE crystal structures. AnCE (cyan)–Ang II (red) complex superposed with human ACE (pink)–Ang II (green) complex (PDB: 4APH) 1. The catalytic zinc ion is shown as a green sphere. Residue labels are in black and pink for AnCE and human ACE proteins respectively. (B) Comparison of Ang II and captopril binding in AnCE. AnCE (cyan)–Ang II (red) complex superposed with AnCE–captopril (yellow) complex (PDB: 2X8Z) [18]. (C) Comparison of Ang II and lisinopril binding in AnCE. AnCE (cyan)–Ang II (red) complex superposed with AnCE–lisinopril (grey) complex (PDB: 2X91) [18].