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 examine three specific aims: (1) What are the structural and energetic bases for the modulation of response regulator function by phosphorylation in CheY and in NarL. NarL regulates the choice of electron acceptor protein the bacterium expresses by acting as an activator of some genes and a repressor of others. The exposure of the DNA binding domain of NarL is regulated by phosphorylation of its response regulator domain, with interactions weakening upon phosphorylation. Conversely, phosphorylated CheY has enhanced affinity for its target FliM. Is there a common structural and mechanism for these distinct outcomes of phosphorylation? (2) What is the structural basis for the modulation of the kinase activity of CheA. CheA forms a hetero-trimeric complex with the transmembrane chemotaxis receptors and the coupling protein CheW. Ligand binding to, and reversible methylation of, the receptor have dramatic effects on CheA activity. What role does CheW play in the coupling mechanism? Where does CheW bind to CheA? What role does CheW play in transmitting the receptor information to CheA? To answer these questions we propose to solve the structures of CheW in complex with CheA using NMR and/or x-ray crystallography. (3) How do the chemotaxis receptors transmit information? The aspartate receptor of E. coil is responsible for chemotaxis to two attractants, aspartate and maltose and for repellent responses to Co (II) ion. Maltose does not bind directly to the receptor but interacts via the periplasmic maltose binding protein (MBP). How does MBP interact with the periplasmic domain of the receptor? Is the strength of that interaction dependent on maltose binding to MBP? Where do CheW and CheA interact with the cytoplasmic domain of the receptor? We propose to use NMR and other physical methods to answer these questions.
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