There is much debate about the role of molecular motions in enzyme catalysis. Previous literature NMR studies have shown that introduction of a distal Serine to Threonine (S99T) mutation in the proly-isomerase Cyclophilin A (CypA) causes a slowdown in a millisecond time scale conformational change that correlates with a decrease in microscopic catalytic rate constants. X-ray crystallography studies suggested that the conformational change implicates a network of coupled side-chain rotations that links Serine99 to active side residues. Because it remains unclear how millisecond timescale motion couple to catalysis, the present work used extensive equilibrium molecular dynamics, umbrella sampling and Markov State (MSM) modeling simulation methodologies to investigate the link between protein motions and function in CypA WT and S99T forms. Comparative analysis of MSMs constructed for the apo CypA WT and S99T forms show that the X-ray crystallography-derived minor state is well reproduced by the simulations. However coupled rotations of the network of side-chains occur at a rate that is at least 3 orders of magnitude faster than suggested by NMR. Further, simulations of substrate-bound WT and S99T show that no coupled side-chain rotations occur during the isomerization reaction. Yet, the simulations derived activation free energies for the cis/trans isomerization reaction support the experimental findings that the mutant is catalytically less active than wild-type. Trajectory analyses show that the increased activation free energies in the S99T-catalyzed reaction are due to decreased probabilities of hydrogen bonding interactions between the transition state and several binding site residues. Decreased hydrogen bonding interactions are shown to be a consequence of a global increase in binding site flexibility due to poorer sidechains packing in the S99T mutant. We therefore conclude that the allosteric effects caused by the S99T mutation are due to decreased electrostatic preorganization of the transition state ensemble, and that previously reported NMR- measured changes in millisecond timescale motions are not causally linked to catalysis.

Allosteric effects in a catalytically impaired variant of cyclophilin A are unrelated to millisecond time scale motions

255th National Meeting (2018)

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