Allosteric effects in cyclophilin mutants may be explained by changes in nano-microsecond time scale motions

Pattama Wapeesittipan,Julien Michel,Malcolm D Walkinshaw,Antonia S J S Mey

doi:10.1038/s42004-019-0136-1

Abstract

This work investigates the connection between stochastic protein dynamics and function for the enzyme cyclophilin A (CypA) in wild-type form, and three variants that feature several mutations distal from the active site. Previous biophysical studies have suggested that conformational exchange between a ‘major’ active and a ‘minor’ inactive state on millisecond timescales plays a key role in catalysis for CypA. Here this hypothesis is addressed by a variety of molecular dynamics simulation techniques. Strikingly we show that exchange between major and minor active site conformations occurs at a rate that is 5 to 6 orders of magnitude faster than previously proposed. The minor active site conformation is found to be catalytically impaired, and decreased catalytic activity of the mutants is caused by changes in Phe113 motions on a ns-μs timescale. Therefore millisecond timescale motions may not be necessary to explain allosteric effects in cyclophilins.

Highlights

This work investigates the connection between stochastic protein dynamics and function for the enzyme cyclophilin A (CypA) in wild-type form, and three variants that feature several mutations distal from the active site
While there is broad consensus that protein motions are implicated in catalysis, there is much debate around the role of conformational changes occurring on a millisecond timescale, and several studies have linked changes in millisecond protein motions with changes in enzymatic function[6,7,8,9]
The focus is on clarifying the nature of protein motions implicated in catalysis for the well-studied enzyme cyclophilin A (CypA)

Summary

Introduction

This work investigates the connection between stochastic protein dynamics and function for the enzyme cyclophilin A (CypA) in wild-type form, and three variants that feature several mutations distal from the active site. Previous biophysical studies have suggested that conformational exchange between a ‘major’ active and a ‘minor’ inactive state on millisecond timescales plays a key role in catalysis for CypA. This hypothesis is addressed by a variety of molecular dynamics simulation techniques. While there is broad consensus that protein motions are implicated in catalysis, there is much debate around the role of conformational changes occurring on a millisecond timescale, and several studies have linked changes in millisecond protein motions with changes in enzymatic function[6,7,8,9]. Fraser et al later used ambient temperature X-ray crystallographic data to determine a high-resolution structure of this CypA state m, revealing an interconversion pathway with the ‘major’ state M that involves coupled rotations of a network of side-chains involving residues

Methods

Results

Conclusion