Structural basis for the C-domain-selective angiotensin-converting enzyme inhibition by bradykinin-potentiating peptide b (BPPb)

Abstract

Angiotensin-converting enzyme (ACE) is a zinc metalloprotease best known for its role in blood pressure regulation. ACE consists of two homologous catalytic domains, the N- and C-domain, that display distinct but overlapping catalytic functions in vivo owing to subtle differences in substrate specificity. While current generation ACE inhibitors target both ACE domains, domain-selective ACE inhibitors may be clinically advantageous, either reducing side effects or having utility in new indications. Here, we used site-directed mutagenesis, an ACE chimera and X-ray crystallography to unveil the molecular basis for C-domain-selective ACE inhibition by the bradykinin-potentiating peptide b (BPPb), naturally present in Brazilian pit viper venom. We present the BPPb N-domain structure in comparison with the previously reported BPPb C-domain structure and highlight key differences in peptide interactions with the S4 to S9 subsites. This suggests the involvement of these subsites in conferring C-domain-selective BPPb binding, in agreement with the mutagenesis results where unique residues governing differences in active site exposure, lid structure and dynamics between the two domains were the major drivers for C-domain-selective BPPb binding. Mere disruption of BPPb interactions with unique S2 and S4 subsite residues, which synergistically assist in BPPb binding, was insufficient to abolish C-domain selectivity. The combination of unique S9–S4 and S2′ subsite C-domain residues was required for the favourable entry, orientation and thus, selective binding of the peptide. This emphasizes the need to consider factors other than direct protein–inhibitor interactions to guide the design of domain-selective ACE inhibitors, especially in the case of larger peptides.

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

“…To date, no structures have been observed with cleaved products where the N-terminal portion is bound solely in the nonprime lobe. We can therefore speculate that the nonprime products are [35]) showing (B) the hole at the base of the nonprime lobe, and (C) highlighting the possible routes for extension of the prime binding lobe. Binding groove/cavity is shown in grey with the active-site zinc as magenta sphere.…”

“…An overlay of the BPPb peptide from a previously published nACE complex [35] with the open nACE structure shows that the BPPb N terminus is positioned approaching the hole in the nonprime lobe (Fig. 11B).…”

“…A at the most flexible regions at the lip of subdomain 1, and the RMSD values for 605 Ca atoms observed in all structures range from 0.14 to 0. 35 A. The active-site zinc ion was coordinated in the usual manner for ACE domains to His-361, His-365 and Glu-389, with its coordination sphere completed by a water molecule (Fig.…”

“…Most of the known substrates of ACE domains are relatively short, around 12 residues or less, and as shown by the 11-mer BPPb bound to cACE and nACE, peptides of this length can fit entirely within the two-lobed binding site [34,35]. However, it has been unclear how longer peptide substrates, such as beta-amyloids, could fit into the active site of ACE.…”

Eje Renina Angiotensina, Enzima Convertidora de Angiotensina 2 y Coronavirus

“…La ECA es un componente esencial del SRAA, producida por el endotelio de los tejidos somáticos constituye una proteína transmembrana con dos dominios catalíticos activos tipo N y C terminales. El clivaje del dominio C terminal genera la carboxipeptidasa soluble que remueve el dipéptido carboxiterminal de AngI, generando AngII, mientras que la hidrólisis de los péptidos vasodilatadores denominados bradiquininas se produce por la acción enzimática de ambos dominios 6 . La ECA2, identificada simultáneamente por Donoghue y Tipnis en el año 2000 7,8 , es una monocarboxipeptidasa homóloga a ECA, con una sola hélice transmembrana, un segmento intracelular y dominios N y C terminales, pero con un único sitio activo enzimático que le da características diferentes a ECA.…”