Studies On The Structure And Dynamics Of Response Regulator RR468

Biomacromolecules,like proteins,are flexible scaffolds assembled from loads of basic building blocks,and are participating in endless dynamic motions,on various time scales,to establish the very fundamentals of life.Liquid NMR technologies can be harnessed to sduty these various dynamic motions at atomic level.This domain of knowledge is sure to reveal more than the structure-activity relationship,but also the dynamics-activity relationship.In this dissertation,RR468,as one of the response regulator receiver domains that widely involved in bacteria two-component signal transduction systems,was studied using solution NMR methods to provide atomic-level information about the structure and dynamics in association with different functional states during the signal transduction cycle.The main molecular response of an receiver domain upon activation is proved to be a metal ion-dependent,phosphorylation-mediated conformation transition.The assembly of the active site produces allosteric structural rearrangement that outreaches distal regions responsible of receiver domain dimerization or interaction with downstream output domains.In this work,the domain-wise modulation in dynamics,by the cooperation of Mg2+and reaction with BeF3-,was determined using CPMG relaxation dispersion and CEST experiments.Results showed that in the free state,RR468 existed as a heterogenous ensemble with fast exchange(>2000 s-1)on both sides of the domain.The addition of 10 times the equivalent of Mg2+slowed the exchange to<500 s-1,and narrowed the exchanging region to the adjacency of the active site.And as revealed by both CEST and CPMG RD,there was clearly a minor state with structure more resembling the fully active state.When reacted with BeF3-and fully activated,the ensemble of RR468 became homogenous,and there could be observed no exchange on the ?s-ms time scale.The allosteric network behind this global dynamics shifts was also investigated.I54A mutation left the ability to coordinate an Mg2+ untouched,but stopped the con-formation transition from propagating to ?4-?4 loop and ?5-?5 loop,thus defied the binding of BeF3-group and activation.M56A mutation uncouples the interaction of?3-?3 loop and the core domain.As a result,residues from ?3-?3 loop were not observable on 1H-15N HSQC,just like the case in free state wildtype RR468,while no ?s-ms time-scale exchange took place in the remaining part of M56A.The solution structure of M56A was also determined using NMR to exclude the possibility of trivial breakdown of the natural fold of receiver domain after mutation.Taken together,154 and M56,also designated D+1 and D+3,with reference to the highly conserved Asp 53,represents two important nodes on the seemingly 'invisible' allosteric network.The atomic-level knowledge of allostery underlying signal transduction and this dynamics-based pattern of rational design of functionally relevant mutation should be helpful to understand the allosteric regulation mechanism that is proved to exists in various families of proteins,which in turn would guide drug design and synthetic biology.