Interaction of hemorphins with ACE homologs

Abstract

Hemorphins, short bioactive peptides produced by enzymatic cleavage of β-hemoglobin, exhibit antihypertensive properties by inhibiting angiotensin-1 converting enzyme (ACE1). ACE1 is a key player in the renin-angiotensin system (RAS) and regulates blood pressure. ACE1 and its homolog, ACE2, which exhibit opposing activities in the RAS, share considerable similarity in their catalytic domains. The primary objective of this study was to identify and contrast the molecular mechanisms underlying the interaction of hemorphins of camels and that of other mammals with the two ACE homologs. In silico docking and molecular dynamics simulations were performed for ACE1 and ACE2, along with in vitro confirmatory assays for ACE1. The C-domain of ACE1, primarily involved in regulating blood pressure, was used along with the N-terminal peptidase domain of ACE2. The findings revealed conserved hemorphin interactions with equivalent regions of the two ACE homologs and differential residue-level interactions reflecting the substrate preferences of ACE1 and ACE2 considering their opposing functions. Therefore, conserved residue-level associations and implications of poorly conserved regions between the two ACE receptors may potentially guide the discovery of selective domain-specific inhibitors. The findings of this study can provide a basis for the treatment of related disorders in the future.

Figure 1

Dose–response curves of camel LVVHem6 and LVVHem6 against ACE1. The data are represented as the mean ± SD of three independent experiments.

Figure 2

Docked confirmation and hydrogen bond interactions of the hemorphin peptides bound to ACE1 and ACE2. (A) Camel LVVHem6 with ACE1 (B) LVVHem6 with ACE1 (C) Camel LVVHem6 with ACE2 (D) LVVHem6 with ACE2. ACE1 and ACE2 are shown in gray cartoon ribbons and their interacting residues are illustrated in a pink stick display; the docked ligand is shown in yellow and orange stick representation, hydrogen bonds are shown as black dashed lines, salt bridges are shown in red, π–π stacking depicted by light green dashed lines, and π-cation stacking is shown in dark green dashed lines.

Figure 3

Root mean square standard deviation (RMSD) of protein Cα atoms obtained from 500 ns simulations. (A) Camel LVVHem6 with ACE1 (B) LVVHem6 with ACE1 (C) Camel LVVHem6 with ACE2 (D) LVVHem6 with ACE2.

Figure 4

Root mean square fluctuation (RMSF) plots obtained from 500 ns simulations. (A) Camel LVVHem6 with ACE1 (B) LVVHem6 with ACE1 (C) Camel LVVHem6 with ACE2 (D) LVVHem6 with ACE2.

Figure 5

Radius of gyration (Rg) of hemorphin peptides from 500 ns simulations. (A) Camel LVVHem6 with ACE1 (B) LVVHem6 with ACE1 (C) Camel LVVHem6 with ACE2 (D) LVVHem6 with ACE2.

Figure 6

Percentage of contact time during which intermolecular polar contacts were retained between ACE1 and ACE2 and hemorphin peptides in the 500 ns systems. (A) Camel LVVHem6 with ACE1 (B) LVVHem6 with ACE1 (C) Camel LVVHem6 with ACE2 (D) LVVHem6 with ACE2. Here, h signifies a hydrogen bond and s signifies a salt bridge.

Figure 7

Percentage of contact time during hydrophobic interactions between ACE1 and ACE2 proteins and hemorphin peptides. (A) Camel LVVHem6 with ACE1 (B) LVVHem6 with ACE1 (C) Camel LVVHem6 with ACE2 (D) LVVHem6 with ACE2.