Gaussian Accelerated Molecular Dynamics Simulations Investigation on the Mechanism of Angiotensin-Converting Enzyme (ACE) C-Domain Inhibition by Dipeptides

Abstract

Angiotensin-converting enzyme (ACE)-inhibitory peptides extracted from food proteins can lower blood pressure by inhibiting ACE activity. A recent study showed that the inhibitory activity of IY (Ile-Tyr, a dipeptide derived from soybean protein) against ACE was much higher than that of LL (Leu-Leu), although they had similar hydrophobic and predicted activity values. It was difficult to reveal the deep molecular mechanism underlying this phenomenon by traditional experimental methods. The Apo and two complex systems (i.e., ACE-LL and ACE-IY) were therefore subjected to 1 μs long Gaussian accelerated molecular dynamics (GaMD) simulations. The results showed that the binding of IY can cause obvious contraction of the active site of ACE, mainly manifested by a significant lateral shift of α13, α14, and α15. In addition, hinge 2 and hinge 3 were more stable in the ACE-IY system, while these phenomena were not present in the ACE-LL system. Moreover, the α10 of the IY-bound ACE kept an inward state during the simulation progress, which facilitated the ACE to remain closed. However, for the LL-bound ACE, the α10 switched between two outward states. To sum up, our study provides detailed insights into inhibitor-induced conformational changes in ACE that may help in the design of specific inhibitors targeting ACE for the treatment of hypertension.

Keywords: Gaussian accelerated molecular dynamics (GaMD) simulations; angiotensin-converting enzyme (ACE); inhibitory peptides; molecular mechanism.

Figure 1

Stereo structures of the ligands and complex: (a) 3D structures of IY and LL. IY is the dipeptide Ile-Tyr, and LL is the dipeptide Leu-Leu; (b) complex of the ACE (angiotensin-converting enzyme) and inhibitory peptide; (c) detailed diagram of the secondary structure of ACE.

Figure 2

The temporal evolution of the RMSDs from their initial structure of three complexes in the region of (a) the whole protein, (b) hinge 1, (c) hinge 2, (d) hinge 3, (e) hinge 4 and (f) the lid. RMSD is root mean square deviation.

Figure 3

(a) RMSFs of Cα atoms in the three systems, and critical regions are highlighted with rectangles. RMSF is root mean square fluctuation; (b) superposition of extracted protein conformations from MD (molecular dynamics) trajectories for critical regions. The Apo means ligand-free protein, LL represents the complex system of protein and dipeptide Leu-Leu, and IY stands for complex system of protein and dipeptide Ile-Tyr.

Figure 4

Analysis of the secondary structural changes of proteins: (a) representative snapshots of the three systems, and hinge 2 and hinge 3 are highlighted with rectangles; (b) DSSP (dictionary of secondary structure of protein) results for the three complexes and the helix probabilities of the corresponding residues. The Apo means ligand-free protein, LL represents the complex system of protein and dipeptide Leu-Leu, and IY stands for complex system of protein and dipeptide Ile-Tyr.

Figure 5

The dynamic cross-correlation map for the 1 μs GaMD simulation trajectories of the three systems. The positive regions are colored in red, whereas the negative regions are shown in blue, representing correlated and anti-correlated motions between residue atoms, respectively. The Apo means ligand-free protein, LL represents the complex system of protein and dipeptide Leu-Leu, and IY stands for complex system of protein and dipeptide Ile-Tyr.

Figure 6

Analysis of the interaction between ACE (angiotensin-converting enzyme) and inhibitory peptides: (a) ligand poses of 10 superimposed structures over 1 μs for IY and LL. IY is the dipeptide Ile-Tyr, and LL is the dipeptide Leu-Leu; (b) time evolution of the RMSDs (root mean square deviations) and (c) corresponding frequencies for LL and IY. LL represents the complex system of protein and dipeptide Leu-Leu, and IY stands for complex system of protein and dipeptide Ile-Tyr; (d) evolution of the number of hydrogen bonds formed between ACE and peptides during MD (molecular dynamic) simulations; (e) average distance between IY and residues interacting with inhibitor.

Figure 7

Schematic diagram of bridging of inhibitors across the active site cleft for (a) ACE–IY and (b) ACE–LL complexes. ACE–IY stands for complex system of protein and dipeptide Ile-Tyr, ACE–LL represents the complex system of protein and dipeptide Leu-Leu.

Figure 8

(a) Active site volume analysis for the three systems: (b) radius of gyration over 1 μs GaMD for the three systems; alignments of (c) LL-bound and (d) IY-bound representative structures to its apo form; the average SASA of (e) α13, (f) α14, and (g) α15 for the three systems. The Apo means ligand-free protein, LL represents the complex system of protein and dipeptide Leu-Leu, and IY stands for complex system of protein and dipeptide Ile-Tyr.

Figure 9

Free energy landscape showing the α10-helix in–out for the complexes (a) ACE–LL and (b) ACE–IY, ACE–LL represents the complex system of protein and dipeptide Leu-Leu, ACE–IY stands for complex system of protein and dipeptide Ile-Tyr; (c) population density of the LL-bound and IY-bound ACE (angiotensin-converting enzyme), D1 was P163@CA-Q308@CA, and D2 was N167@CA-L175@CA. The respective global minimum structures for both structures are also shown below.

Figure 10

Two-dimensional free energy landscapes (FELs) created by projecting the principal components PC1 (principal component 1) and PC2 (principal component 2) for (a) Apo, (b) ACE–IY, and (c) ACE–LL; the representative structures of (d) Apo, (e) ACE–IY, (f) ACE–LL from cluster analysis are shown in the below panel. Apo means ligand-free protein, ACE–IY stands for complex system of protein and dipeptide Ile-Tyr, ACE–LL represents the complex system of protein and dipeptide Leu-Leu.

Figure 11

Interactions affecting the active site in ACE upon binding of LL and IY. The hydrogen bonding between different residues, such as (a) S284–E376, (b) D377–T372, and (c) T372–E162, are shown; the schematic diagrams highlighting the critical interactions for (d) Apo, (e) ACE–LL, and (f) ACE–IY. Apo means ligand-free protein, ACE–LL represents the complex system of protein and dipeptide Leu-Leu, ACE–IY stands for complex system of protein and dipeptide Ile-Tyr.

Figure 12

Contour plots of free energies as a function of the backbone angles of φ and ψ for the residues (a) E284, (b) E376, (c) D377, (d) T372, and (e) E162 of the ACE–LL and ACE–IY complexes. Apo means ligand-free protein, ACE–LL represents the complex system of protein and dipeptide Leu-Leu, ACE–IY stands for complex system of protein and dipeptide Ile-Tyr.