Mechanistic Studies of Bacterial Chemotaxis

University Of California Santa Barbara, Santa Barbara CA

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Abstract

Bacterial chemotaxis is one of the best understood signaling systems in biology. It is one of a large number of "two component" sensory systems in bacteria that use two proteins, a histidine auto kinase (CheA) and the response regulator proteins (CheY and CheB) that are the kinase substrates that are phosphorylated on aspartate residues. Phosphorylation of the response regulator domain modulates its interactions with its target domain(s) resulting in increased or decreased affinity for the other domain, depending on the system. We propose to use modern nuclear magnetic resonance techniques and other physical methods to answer questions in two specific aims: (1) What is the structural basis for the modulation of the kinase activity of CheA. The rate-limiting step for CheA activity involves autophosphorylation of a histidine residue on a distinct histidine phosphotransfer domain, Hpt, by ATP at the catalytic domain of CheA. After its phosphorylation, the Hpt domain must transfer its phosphate to the kinase substrate proteins CheY and CheB. Where does the Hpt domain reside and how does it gain access to the catalytic domain? What are the motional properties of the Hpt domain in the resting and phosphorylated state? CheA forms a hetero-trimeric complex with the transmembrane chemotaxis receptors and the coupling protein CheW. How do these three proteins interact and how do events at the receptor modulate CheA activity. (2) Phosphorylated CheY, produced by CheA, modulates the sense of rotation of the bacterial rotary flagellar motors. The first step in the process is the binding to a recognition sequence in FliM, a protein of the "switch complex " made up of three proteins (FliM, FliG and FliN) located on the rotor of the motor. How does this binding event result in a change in the sense of rotation of the motor? How does the CheY-FliM complex communicate this information to the rest of the components of the motor?