Allosteric effects in catalytic impaired variants of the enzyme cyclophilin A may be explained by changes in nano-microsecond time scale motions

Abstract

There is much debate about the mechanisms by which molecular motions influence catalysis in enzymes. This work investigates the connection between stochastic protein dynamics and function for the enzyme cyclophilin A (CypA) in wild-type (WT) form, and three variants that features several mutations that are distal from the active site. Previous biophysical studies have suggested that conformational exchange between a major active and a minor inactive state on millisecond time scales plays a key role in catalysis for CypA. Here this hypothesis was addressed by a variety of molecular dynamic (MD) simulation techniques. The simulations reproduce X-ray crystallography derived evidence for a shift in populations of major and minor active site conformations between the wild-type and mutant forms. Strikingly, exchange between these active site conformations occurs at a rate that is 5 to 6 orders of magnitude faster than previously proposed. Further analyses indicate that the minor active site conformation is catalytically impaired, and that decreased catalytic activity of the mutants may be explained by changes in Phe113 motions on a ns-s time scale. Therefore previously described millisecond time scale motions may not be necessary to explain allosteric effects in CypA mutants.