Microsecond timescale MD simulations at the transition state of HMGR predict remote allosteric residues.

Understanding the mechanisms of enzymatic catalysis requires a detailed understanding of the complex interplay of structure and dynamics of large systems that is a challenge for both experimental and computational approaches. More importantly, the computational demands of QM/MM simulations mean that the dynamics of the reaction can only be considered on a timescale of nanoseconds even though the conformational changes needed to reach the catalytically active state happen on a much slower timescale. Here we demonstrate an alternative approach that uses transition state force fields (TSFFs) derived by the quantum-guided molecular mechanics (Q2MM) method that provides a consistent treatment of the entire system at the classical molecular mechanics level and allows simulations at the microsecond timescale. Application of this approach to the second hydride transfer transition state of HMG-CoA reductase from (HMGR) identified three remote residues, R396, E399 and L407, (15-27 Å away from the active site) that have a remote dynamic effect on enzyme activity. The predictions were subsequently validated experimentally site-directed mutagenesis. These results show that microsecond timescale MD simulations of transition states are possible and can predict rather than just rationalize remote allosteric residues.