Freedom in Transition: Computational Studies of Escherichia coli MTAN and Vibro cholerae MTAN

Abstract

Transition state analogue inhibitors of Escherichia coli 5'-methylthioadenosine/S-adenosylhomocysteine nucleosidase (EcMTAN) and Vibrio cholerae 5'-methylthioadenosine/S-adenosylhomocysteine nucleosidase (VcMTAN) are among the tightest-binding noncovalent inhibitors known. Closely mimicking the transition state of the substrate molecule, these inhibitors disrupt quorum sensing pathways in E. coli and V. cholerae. Despite the similarities of these two homologous enzymes' static active site structures, experimental studies detected a thousand-fold difference in their affinity the transition state analogue butylthio-DADMe-Immucillin-A (BDIA), suggesting protein dynamics may play a role in their differential binding. To elucidate the role of dynamics, we conducted classical molecular dynamics (MD) simulations of VcMTAN and EcMTAN in complex with BDIA. The small differences in electrostatic and hydrophobic interactions detected in the two systems are unlikely to account for the large difference in the affinity of the enzymes for BDIA. Instead, the greater conformational freedom and flexibility exhibited by the EcMTAN--BDIA system suggests that transition state analogues that preserve protein dynamic motions related to the formation of the transition state are the ones that bind more tightly. To determine the exact role of dynamics in the formation of the transition state in the two enzymes, we conducted transition path sampling (TPS) simulations of their hydrolysis of 5'-methylthioadenosine (MTA). Using a novel mechanism for generating the initial reactive trajectory, TPS has generated one hundred reactive trajectories for both VcMTAN and EcMTAN. A subset of these trajectories was selected for committor analysis, which can identify the slices closest to the transition state. Committor analysis confirmed that the mechanism of VcMTAN's hydrolysis of MTA is nucleophilic substitution by electrophile migration.