Mechanisms Of Transmembrane Signaling By Bacterial Chemoreceptors: Studies Of Dynamics And Cofactor Reactivity

Flagellated bacteria constantly and actively search for optimum niches for their survival and proliferation by swimming in the environment. A family of receptor proteins, named chemoreceptors or methyl-accepting chemotaxis proteins (MCPs), along with cellular energy sensor Aer and sensory rhodopsins (SR) guide the cells towards optimal chemical and spectral compositions by controlling the activity of intracellular kinase CheA and thereby regulating cellular taxes (or movements). These receptors also serve as model systems for transmembrane signal transduction and cell-environment interaction. Although the receiver domains in these receptors vary greatly in sequence, function, and even in disposition relative to the membrane, the cytoplasmic domains of all of these transmembrane proteins share high homology and signal through the same kinase CheA. We engineered two variants of the cytoplasmic domain of E. coli aspartate receptor Tar, H1-Tar and H1-2-Tar, that mimic the ligand unbound and bound states of the full length native receptor, respectively, in order to uncover the general principle behind kinase activity regulation. As translated, H1-Tar stimulates the kinase, whereas H1-2-Tar deactivates the kinase both in vivo and in vitro, despite similar binding to CheA. These variants also respond to the modifications in four conserved glutamate and glutamine residues as the native Tar: mutation of two glutamines to glutamates renders H1-Tar deactivating, whereas H1-2-Tar stimulates the kinase upon mutation of the two glutamates to glutamines. Continuous wave and pulsed dipolar electron spin resonance spectroscopic studies of the spin labeled variants reveal dynamical coupling throughout the cytoplasmic domain of MCPs and that dynamics in the two ends of the domain are inversely correlated. The dynamics in the membrane distal region of the domain, named protein interaction region (PIR) that interacts with the kinase via an adaptor protein CheW correlate with the activating state of the receptors: conformationally dynamic PIR deactivates CheA and relatively static PIR activates the kinase. Thus, we find that these receptors regulate the kinase activity based on the receiver domain state and modifications in those conserved residues by altering the dynamics throughout the cytoplasmic domain. Of these receptor proteins, the state of the receptive PAS domain of the energy sensor Aer relevant for CheA activity modulation is not particularly well described. We have, for the first time, purified bacterial Aer and have confirmed that Aer PAS domain binds FAD as a cofactor. In vitro sodium dithionite treatment reversibly reduces FAD in Aer from fully oxidized state to the single electron reduced anionic semiquinone (ASQ) state supporting the notion that Aer FAD samples different redox states to modulate the kinase activity. We show that Aer in fully oxidized FAD state activates the kinase, whereas in ASQ state inhibits the kinase. These results suggest that Aer senses the electron flow in the electron transport chain through FAD bound at the PAS domain and modulates the kinase activity based on the FAD redox state. Overall, this dissertation gains insights into the mechanisms of how these transmembrane receptors regulate the kinase activity in order to govern bacterial motion in response to the environmental stimuli