Molecular dynamics simulation perspective on the role of protein motions in allosteric regulation
A deep understanding of the physicochemical principles that underpin allosteric regulation in proteins is a major objective of contemporary biophysical chemistry research. This work focuses on understanding protein allostery mechanisms by means of computer simulations. Numerical studies and molecular dynamics (MD) simulation were used to investigate allosteric effects using discrete and continuous energy landscape representation of simple generic protein models in the framework of the recently proposed ensemble allostery model. Efforts turned next to the elucidation of specific allosteric effects in Cyclophilin A (CypA), an enzyme that catalyzes cis/trans isomerisation of amide bonds in Proline residues. There has been a vigorous debate about the functional role of protein motions in this enzyme. Previous studies have proposed a causal link between specific millisecond time-scale motions and catalysis in CypA. X-ray crystallography and NMR measurements suggest that several mutations remote from the catalytic site of CypA cause changes in populations and rates of exchanges between a ‘major’ and a ‘minor’ conformational state, that correlates with decreased catalytic activity of mutants with respect to the wild-type form. Atomistic molecular dynamics simulations of CypA were carried out to investigate further the connection between rates of protein motions and catalysis. Markov state models (MSM) of the enzymes in apo form were constructed to estimate rates of transition between conformational states. The MSM-derived structures and populations of the simulated conformational states reproduced well the X-ray crystallography data. Remarkably the rate of exchanges between the computed conformational states were 5 to 6 orders of magnitude faster (ns-μs timescale) than the rates reported from NMR measurements (ms-s timescale). Umbrella sampling MD techniques were then used to compute free energy profiles for the cis/trans-isomerization reaction of CypA in the computed ‘major’ or ‘minor’ conformational states. The results show that in all forms of CypA the ‘major’ conformational state is catalytically competent, whereas the ‘minor’ conformational state is functionally inactive due to poorer hydrogen bonding interactions with the transition state substrate. Finally, a model for catalysis in CypA was constructed by combining the results from the MSM and Umbrella Sampling simulations, leading to overall calculated isomerization rates consistent with experimental measurements. Therefore, it can be concluded that changes in fast (ns-μs) time scale motions are sufficient to explain the reduction of catalytic activity of CypA mutants.