Sybyl version 8

The models for both C- and N-domains of ACE were derived from the Protein Data Bank (http://www.rcsb.org) structures having PDB codes 4APH and 4BZS, respectively. Protein structures and ligands were processed by using the software Sybyl, version 8.1 (www.tripos.com, Certara USA, Inc., Princeton, NJ, USA). All atoms were checked for atom- and bond-type assignments. Amino- and carboxyl-terminal groups were set as protonated and deprotonated, respectively. Hydrogen atoms were computationally added to the protein and energy-minimized using the Powell algorithm whit a coverage gradient of ≤0.5 kcal (mol Å)–1 and a maximum of 1500 cycles.
Anatomy of the pocket. The two catalytic domains of ACE originated from tandem gene duplication (ref) and maintain the same 3D organization with 51% of sequence identity (according to global alignment by using the Needleman–Wunsch algorithm; http://www.ebi.ac.uk/Tools/psa/emboss_needle). Both domains hold a huge pocket with similar shape which crosses the entire protein body. However, analyses were focused on catalytic sites retracing the mode of action of inhibitory drugs (Yates et al., 2014). The regions lining the catalytic site maintain the same organization in both domains and both pockets share a prevalently hydrophobic environment, albeit they differ for 7 amino acid substitutions.