Structural Basis For Substrate Specificity And Domain Mobility In Ace - Open For Business

Objective: Angiotensin-1-converting enzyme (ACE) is a zinc metallopeptidase that is well known for its role in cardiovascular physiology. Through its cleavage of peptides angiotensin I and bradykinin, ACE is involved in the renin-angiotensin-aldosterone and kinin systems, respectively. However, ACE also catalyses the cleavage of a variety of substrates, and thus participates in numerous other physiological functions. The aim of this study was to investigate the hinging mechanism of ACE and the molecular basis for its hydolysis a wide range of substrates with exo- and endopeptidase activity. Design and method: Minimally glycosylated N- and C-domain human ACE proteins (N389 and G13 respectively) and N389- S2-Sprime-nACE were produced by expression in cultured mammalian CHO cells and purified to homogeneity. ACE domains were crystallised and X-ray diffraction data were collected at the Diamond Light Source. Initial phases for the native structure were obtained by molecular replacement with PHASER. Results: In the present study, we present the high resolution crystal structures of native and S2-Sprime mutant ACE N-domains in an open conformation, and a closed C-domain structure where the C-terminus of a symmetry-related molecule is observed inserted into the active site cavity and binding to the zinc ion. Conclusions: Human ACE hydrolyzes a wide range of peptide substrates of varying lengths with carboxypeptidase, and endopeptidase activity. The crystal structures presented in this study shed light on how the ACE domains structurally achieves this diversity of activities. We have crystallized the first open nACE structure, demonstrating that ACE opens like ACE2 to give a wide groove that allows peptides to easily access the binding site. The shape of the groove encourages peptides to adopt an extended linear conformation, and there are likely to be interactions starting near the edge of the groove that are specific for the different peptide substrates that orientate and position them for the different types of hydrolysis. The cACE structure, showing C-terminal insertion from a symmetry related molecule, explains how sACE is capable of cleaving long peptides that are too big to be enclosed within the non-prime binding lobe.