Study On Thermostability Of The Cytosolic Creatine Kinase Using Molecular Dynamics Simulations

Creatine kinase(CK)catalyzes the reversible phosphotransfer between ATP and phosphocreatine,and is thus responsible of maintaining homeostasis of intracellular ATP level.Human cytosolic CK contains two isoforms,the muscle-type(M)and the brain-type(B),which frequently function as homodimers.Despite high similarity in amino acid sequences and tertiary structures,hBBCK and hMMCKexhibit significantly different thermostabilities.Their molecular mechanism of thermal inactivation is supposed to be related to the subunit dissociation at the dimeric interface.Further experiments showed that one single residue mutation at position 36(Pro in hBBCK vs.Leu in hMMCK)can eliminatethe difference in thermostabilities between them,but the molecular mechanism remains unknown.In this thesis,we engaged the molecular dynamics simulations to preliminary explore the factors influencing the thermostabilities difference between CKs,and to in-depth study the mechanism accounting for the difference in thermostabilities between hBBCK and hMMCK.Firstly,the intrinsic properties of hBBCK and hMMCK were compared.hMMCK shows better surface hydrophilicity,more internal hydrogen bonds and salt bridges,indicating more thermostable than hBBCK.However,the dimeric interface of hMMCK is not more stable than hBBCK.In Gaussian network modelanalysis,residue 36 in hBBCK interacts with N-terminal domain in the other chain more easily whencompared with that in hMMCK.They exhibit similar fluctuations under high temperature simulations and the difference mainly occurs in the linking loop between N-terminal domain and C-terminal domain.The dissociation of dimer was not observed during 20 ns extra-high-temperature simulations,and thus need further studies by other accelerated methods.Secondly,we conclude that residue 36 accounts for the thermostability of human cytosolic CK by changing the binding affinity of dimeric interface based on free energy calculations of simulations under normal temperature.The mechanism of how residue 36 affects dimer dissociation was further studied by functional mode analysis and network analysis.As compared to Pro36,Leu36 can connectmore local residues by forming physical contacts and collective movements,and simultaneously strengthen the whole intra-chain connection,which suppresses the movement amplitude of interface residues along the separating direction and eventually inhibits the dimer dissociation.Meanwhile,accelerated molecular dynamics simulations were performed to observe the dissociation of dimer interface directly.However,despite the large conformational change in hBBCK,dimer dissociation did not happen during 200 ns simulations.Further analysis shows that the large conformational change in hBBCK was caused mainly by the relative movement of two chains,which prevents substrates entering the active site.Therefore,the conformational change of active site may also contribute to the thermostabilities difference between hBBCK and hMMCK besides dimer dissociation.Finally,this thesis briefly describes the other involved research that is the Na+ /K+ selectivity mechanism of eukaryotic voltage-gated sodium channels based on the crystal structure of prokaryotic sodium channel NavRh.