Mechanism and energetics of phosphorylation-mediated signaling

Two-component systems dominate signal transduction in bacteria, plants, and lower eukaryotes. In response to an environmental stimulus, signaling is accomplished by the transfer of a phosphoryl group from a conserved histidine in the transmitter domain of the histidine kinase to a conserved aspartate in the receiver domain of the response regulator. Phosphorylation of receiver domains facilitates their regulation of downstream output domains or proteins to elicit a cellular response. The goal of this dissertation is to investigate the mechanism by which phosphorylation mediates signaling in these molecular switch proteins using NMR spectroscopy.; Tandem connections of His-Asp signaling modules form highly evolved pathways termed a phosphorelay. To address whether the function of phosphotransfer has a distinct structural basis of activation when compared the function of regulation exhibited by conventional response regulators, the NMR solution structure of the first response regulator from a phosphorelay was solved in its activated state (Chapter 1). Structural perturbations in sporulation stage 0 F (Spo0F) were observed in a new region distal from the common signaling surface and can be rationalized by forming the interface with its immediate downstream target.; Previous NMR dynamics studies on different functional forms of the receiver domain of Nitrogen regulatory protein C (NtrC) in conjunction with varying amounts of biological activity suggested that activation was achieved through a pre-existing equilibrium by which mutation partially and phosphorylation fully shifts the equilibrium of the inactive and active states towards the active form. An investigation of the energy landscape using novel combinations of quantitative NMR relaxation experiments (Chapter 2) was conducted to elucidate the mechanism of a population shift by mutation and phosphorylation in NtrC (Chapter 3). The mechanism of activation by mutation was a destabilization of the inactive state whereas a phosphoryl analogue was found to bind only the lowly populated active state which rationalizes the requirement for a pre-existing equilibrium and exemplifies the basis of allostery.; The role of a conserved hydroxyl containing residue poised for communication between the active site of phosphorylation and the allosteric site was investigated in the allosteric network in the receiver domain of NtrC (Chapter 4). Mutation of T82 introduced into wild-type and a partially active D86NA89T mutant allowed for probing of the allosteric network in a bi-directional manner. T82V in NtrC could not be activated in the presence of a phosphoanalogue and upon incorporation into a partially active mutant, caused a shift of the equilibrium towards the inactive state with a subsequent loss of constitutive activity. T82V was found to uncouple the intradomain allosteric network in the receiver domain of NtrC.