Angiotensin‐converting enzyme open for business: structural insights into the subdomain dynamics

Abstract

Angiotensin-1-converting enzyme (ACE) is a key enzyme in the renin-angiotensin-aldosterone and kinin systems where it cleaves angiotensin I and bradykinin peptides, respectively. However, ACE also participates in numerous other physiological functions, can hydrolyse many peptide substrates and has various exo-and endopeptidase activities. ACE achieves this complexity by containing two homologous catalytic domains (N-and C-domains), which exhibit different substrate specificities. Here, we present the first open conformation structures of ACE N-domain and a unique closed C-domain structure (2.0 A) where the C terminus of a symmetry-related molecule is observed inserted into the active-site cavity and binding to the zinc ion. The open native N-domain structure (1.85 A) enables comparison with ACE2, a homologue previously observed in open and closed states. An open S 2 _S 0-mutant N-domain structure (2.80 A) includes mutated residues in the S 2 and S 0 subsites that effect ligand binding, but are distal to the binding site. Analysis of these structures provides important insights into how structural features of the ACE domains are able to accommodate the wide variety of substrates and allow different peptidase activities. Database The atomic coordinates and structure factors for Open nACE, Open S2_S 0-nACE and Native G13-cACE structures have been deposited with codes 6ZPQ, 6ZPT and 6ZPU, respectively, in the RCSB Protein Data Bank, www.pdb.org

ACE2 and ACE: structure-based insights into mechanism, regulation and receptor recognition by SARS-CoV

“…The first crystal structures of the ACE2 peptidase domain were reported as an open native structure and a closed conformation in complex with the inhibitor MLN-4760 [84]. In contrast, the majority of ACE structures solved to date have shown the domains in a closed conformation with the open conformation only recently elucidated [91]. Crystal structures of the ACE and ACE2 peptidase domains in the closed conformation (Figure 2A-C) indicate that the overall fold of these domains is retained [65,66,84], in line with the high degree of sequence conservation between them.…”

“…The tertiary structure of ACE shows the ellipsoid, divided into two approximately equal-sized subdomains (1 and 2) (Figure 2A). The subdomains are not simply the N-and C-terminal halves of the protein, instead the polypeptide chain crosses between the two subdomains five times (subdomain 1: [91] and ACE2 [84] retain the same secondary structure elements and show that the two subdomains open in a clam-shell like manner for both enzymes, resulting in a deep groove leading to the active site at the base (Figure 2E,F). In the closed structures, the two subdomains are tightly connected allowing limited access into the active site and the deep groove is replaced by a two-lobed binding cavity with the active site zinc ion coordinated at the narrowest point (Figure 2B,C).…”

“…The base of this 'lid-like' region is adjacent to a hole that leads into the non-prime lobe of the binding cavity. This may have implications for the ability of these ACE domains to cleave peptides that are too long to fit into the binding cavity, which is described in more detail below [91].…”

“…However, both ACE and ACE2 are capable of cleaving much longer substrates such as amyloid-β peptides (ACE) and apelin-36 (ACE2), both of which are over 35 residues in length [13,14,100]. It has been unclear how these could be accommodated and cleaved by these domains, but a recent native C-domain structure has shed light on a possible explanation [91]. A small hole in the non-prime lobe adjacent to the flexible 'lid-like' region has multiple conformations in the N-domain structures thereby altering the size of the hole.…”

“…Both domains of ACE and the ACE2 peptidase domain have conserved 'lid-like' regions and possess this hole. In the recent C-domain structure [91], the C-terminus of a symmetry related cACE molecule from the crystal lattice is shown to insert through this hole, extend across the non-prime lobe, and coordinate with the active site zinc ion. Therefore, it was suggested that long peptides could be accommodated by the N-terminal portion passing out through this hole and occupy space outside the ACE domain.…”