RESUMO
Angiotensin-1-converting enzyme (ACE) is a zinc metalloprotease that plays a major role in blood pressure regulation via the renin-angiotensin-aldosterone system. ACE consists of two domains with differences in inhibitor binding affinities despite their 90% active site identity. While the C-domain primarily controls blood pressure, the N-domain is selective for cleavage of the antifibrotic N-acetyl-Ser-Asp-Lys-Pro. Inhibitors, such as 33RE, that selectively bind to the N-domain thus show potential for treating fibrosis without affecting blood pressure. The aim of this study was to elucidate the molecular mechanism of this selectivity. ACE inhibition by 33RE was characterized using a continuous kinetic assay with fluorogenic substrate. The N-domain displayed nanomolar (Ki = 11.21 ± 0.74 nm) and the C-domain micromolar (Ki = 11 278 ± 410 nm) inhibition, thus 1000-fold selectivity. Residues predicted to contribute to selectivity based on the N-domain-33RE co-crystal structure were subsequently mutated to their C-domain counterparts. S2 subsite mutation with resulting loss of a hydrogen bond drastically decreased the affinity (Ki = 2 794 ± 156 nm), yet did not entirely account for selectivity. Additional substitution of all unique S2 ' residues, however, completely abolished selectivity (Ki = 10 009 ± 157 nm). Interestingly, these residues do not directly bind 33RE. All mutants were therefore subjected to molecular dynamics simulations in the presence and absence of 33RE. Trajectory analyses highlighted the importance of these S2 ' residues in formation of a favourable interface between the ACE subdomains and thus a closed, ligand-bound complex. This study provides a molecular basis for the intersubsite synergism governing 33RE's 1000-fold N-selectivity and aids the future design of novel inhibitors for fibrosis treatment. ENZYMES: Angiotensin converting enzyme (ACE, EC 3.4.15.1).