ACE-domain selectivity extends beyond direct interacting residues at the active site

Area of Science:

• Biochemistry
• Enzymology
• Structural Biology

Background:

• Angiotensin-converting enzyme (ACE) regulates blood pressure via angiotensin II formation.
• ACE possesses two catalytic domains (nACE and cACE) with distinct substrate and inhibitor affinities.
• Understanding domain-specific interactions is key for developing selective ACE inhibitors.

Purpose of the Study:

• To investigate unique properties of nACE inhibitor backbones and their interaction with nACE hinging.
• To elucidate the role of specific nACE residues in inhibitor binding and selectivity.
• To explore a general mechanism of ACE inhibition involving synergistic subsite and interface interactions.

Main Methods:

• Kinetic analysis of nACE residue mutations.
• Determination of high-resolution crystal structures of mutated nACE complexed with inhibitors.
• Affinity measurements for nACE-selective and non-domain-selective inhibitors.

Main Results:

• Mutation of unique nACE residues (S2 pocket, S' subsites) decreased affinity for nACE-specific inhibitors.
• Combined S2_S' mutations abrogated nACE-selectivity for specific inhibitors, but not non-selective ones.
• Structural data confirmed distal residue involvement and synergistic effects in inhibitor binding.

Conclusions:

• nACE-selective inhibitor affinity depends on both direct binding site interactions and distal residue effects.
• A general ACE inhibition mechanism involves synergistic interactions across subsites and the sub-domain interface.
• These findings offer insights into stabilizing active site loops for enhanced inhibitor binding.