Cysteine-mediated dynamic hydrogen-bonding network in the active site of Pin1.
Enzymes catalyze a plethora of chemical reactions that are tightly regulated and intricately coupled in biology. Catalysis of phosphorylation-dependent cis-trans isomerization of peptidyl-prolyl bonds, which act as conformational switches in regulating many post-phosphorylation processes, is considered to be one of the most critical. Pin1 is a cis-trans isomerase of peptidyl-prolyl(ω-) bonds of phosphorylated-Ser/Thr-Pro motifs and has been implicated in many diseases. Structural and experimental studies are still unable to resolve the mechanistic role and protonation states of two adjacent histidines (His59 and His157) and a cysteine (Cys113) in the active site of Pin1. Here, we show that the protonation state of Cys113 mediates a dynamic hydrogen-bonding network in the active site of Pin1, involving the two adjacent histidines and several other residues that are highly conserved and necessary for catalysis. We have used detailed free energy calculations and molecular dynamics simulations, complementing previous experiments, to resolve the ambiguities in the orientations of the histidines and protonation states of these key active site residues, details that are critical for fully understanding the mechanism of Pin1 and necessary for developing potent inhibitors. Importantly, Cys113 is shown to alternate between the unprotonated and neutral states, unprotonated in free Pin1 and neutral in substrate-bound Pin1. Our results are consistent with experiments and provide an explanation for the chemical reactivity of free Pin1 that is suggested to be necessary for the regulation of the enzyme.
Department of Chemistry and the Center for Biotechnology and Drug Design, Georgia State University , Atlanta, Georgia 30302-4098, United States.
Arghya
A
Barman
Donald
D
Hamelberg
D000595:Amino Acid Sequence; D055162:Biocatalysis; D020134:Catalytic Domain; D017124:Conserved Sequence; D003545:Cysteine / Q000737:chemistry / Q000378:metabolism*; D030562:Databases, Protein; D006639:Histidine / Q000737:chemistry / Q000378:metabolism; D006801:Humans; D006860:Hydrogen Bonding; D008958:Models, Molecular; D056004:Molecular Dynamics Simulation; D000072340:NIMA-Interacting Peptidylprolyl Isomerase; D009842:Oligopeptides / Q000737:chemistry / Q000378:metabolism*; D019696:Peptidylprolyl Isomerase / Q000737:chemistry / Q000378:metabolism*; D010766:Phosphorylation; D011485:Protein Binding; D011487:Protein Conformation; D011499:Protein Processing, Post-Translational; D011522:Protons; D015203:Reproducibility of Results; D013816:Thermodynamics
D000072340:NIMA-Interacting Peptidylprolyl Isomerase; D009842:Oligopeptides; D011522:Protons; D006639:Histidine; C000606846:PIN1 protein, human; D019696:Peptidylprolyl Isomerase; D003545:Cysteine