Evaluating the protonation state of the catalytic Cys25 in cruzain cysteine protease: A target for Chagas disease.

Cruzain (Cz), the major cysteine protease of Trypanosoma cruzi, the etiological agent of Chagas disease, employs Cys25 as its catalytic nucleophile, enabling peptide bond hydrolysis via nucleophilic attack on the carbonyl carbon of substrates. The pKa of Cys25 can be modulated by the local environment in the free enzyme or upon formation of pre-reactive complexes with substrates or inhibitors. Here, we employ molecular dynamics (MD) simulations, free energy calculations, and constant-pH simulations with explicit solvent to investigate the protonation state of Cys25 in the apoenzyme and in complexes with either a substrate mimic (Ac-Ala-Ala-Ala-Gly-Ala-OCH₃) or the covalent inhibitor K777 (N-methyl-piperazine-phenylalanyl-homophenylalanyl-vinylsulfone-phenyl). The simulations consistently support the presence of a neutral Cys25/His162 dyad across all states examined. Binding of either substrate or inhibitor reinforces a weak hydrogen bond between Cys25 and His162 but does not substantially perturb the dyad's protonation state. These findings suggest that cruzain's catalytic dyad remains predominantly neutral in the apo and bound forms, with the formation of a CysS/HisH ion pair likely occurring as an early event during catalysis or covalent inhibition. These insights have important implications for understanding the catalytic mechanism and for the rational design of cruzain-targeted therapeutics.