핵자기공명학을이용한M.tuberculosisAK와그변이체의기질결합부위에대한구조연구

Abstract

Adenylatekinase(AK)는ATP,AMP기질과결합하고Mg2+금속이온을매개로하여고에너지인산화작용을통해2개의ADP를생성하는촉매효소(Mg2+‧ATP+AMP⟷Mg2+‧2ADP)로써,물질의에너지대사와nucleotide합성에관여한다.진핵생물의AK1과결핵균(Mycobacteriumtuberculosis)의AK(AKmt)는아미노산서열에서서로유사성을갖지만,AKmt가AK1대비약10%정도의낮은효소활성을갖는다.AK1를구성하는요소중하전을갖는아미노산이AK의효소활성과기질친화력에영향을주는것을참고하여유전자조합과세포배양을통해AKmt의LIDdomain중음전하를나타내는Glu[122]를양전하를나타내는lysine으로변형시킨singlemutant(AKmtE122K)와같은방법으로122번과p-loop의non-polarAla[11]을polar한serine으로변형시킨doublemutant(AKmtDM)를만들어AK1과부분적으로동일한아미노산서열을갖도록하였으며,효소활성을비교한결과기존대비12배이상높은활성을나타냈다.AKmtDM의기질결합을이해하기위해micro-titration을수행하였고결합전․후다핵-다차원펄스NMR을이용하여신호지정을하여반응성을추적하였다.이러한활성변화의규명을위해단백질의변이가기질결합전․후효소의운동성에미치는영향과촉매활성증가의상관관계를NMR데이터와분자동역학실험을수행하여구조적메카니즘연구를진행하였다. AKmt와AKmtDM의chemicalshiftperturbation분석결과Lys[122]주위의residues와LIDdomain의변화를확인할수있었으며α3_helixsite의말단부분에해당하는Asp[54]의perturbation으로부터AMPbindingdomain과의inter-communication을예측할수있었다.AKmt과AKmtDM의15NT1,T2,15N-1Hhetero-nuclearNOErelaxation실험을통해rotationalcorrelationtime()이각각10.725ns와11.104ns로AKmt가상대적으로compact한구조를가짐으로써분자운동성이낮음을알수있었다.Relaxationdata를이용한model-free분석결과AKmt와AKmtDM의각residues의orderparameter차이로부터,ATPliddomain의Gly[128],Asp[131],Asp[132]와AMPbindingdomain의α3_helixsite말단의Arg[51],Asp[54],Ala[55],Gly[56],Asp[57]와ATP또는AMP기질말단의phosphategroup이밀접하게결합할것으로예상되는Glu[33],Ile[39],Glu[44]의flexibility가증가함을알수있었다.GROMACS를이용한10ns분자동역학실험과PCA분석으로부터ATPbindingdomain의mutation이단백질내inter-communication을안정하게하여AMPbindingdomain의운동성을증가시켜AMP와의기질결합력을증가시킬것으로판단된다. ATP와AMP기질적정실험으로부터chemicalshiftperturbation분석결과단백질모두ATPsaturation상태에서Gly[32],Gly[85]와같은AMPbindingdomain의residues에0.2ppm이상의강한perturbation을확인할수있었다.기질결합영역의bindingdomainperturbation결과ATPbindingdomain과AMP기질결합의값은15N과1H각각0.928과0.910로상관성이떨어짐을알수있었고기질결합에따른방향성분석으로부터AMP의ATPbindingdomain결합이ATP결합을저해하는것과는달리ATP의결합은AMPbindingdomain의운동성에영향을미쳐단백질의AMP기질결합을증가시킬것으로예상되었다. Dissociationconstant()분석결과ATPbindingdomain에서의ATP결합력이AKmtDM는:73μM으로AKmt의:498μM보다6.82배높은결합력을보였다.또한AKmt는ATPsaturation후AMP를결합하였을때AMP를단독으로결합했을때보다약7.6배낮은748μM의를나타냈으며,AKmtE122K는4.6배,AKmtDM은약4배의AMP결합증가를나타냈다. Backbonedynamics분석과분자동역학실험으로부터AKmtDM의Glu[118]과Lys[122]의정전기적상호작용과p-loop의수소결합네트워크를통해기질결합및효소활성에중요한역할을하는Arg[123]을구조적으로안정화시킴으로써분자인지기능을향상시켜ATP기질결합력을높이고결합된ATP는AMPbindingdomain과구조적인상호작용을통해AMP기질의결합을가속화시킴으로써AKmt의효소활성을증가시키는것을확인할수있었다.