The overall objective of the project is to determine the mechanism by which bacterial chemotaxis receptors regulate the activity of the central kinase CheA. Bacterial chemotaxis is a well-studied two-component signaling system and is also essential for infection by some pathogens. Two-component signaling systems are widespread in prokaryotes but not found in mammals, making chemotaxis proteins potential targets for novel antibiotics. The specific objective is to determine the molecular details of the interaction between chemoreceptors and CheA, and how these change to control kinase activity. Chemoreceptors function within large membrane-bound hexagonal arrays of receptors, CheA, and CheW.
Aims 1 -2 will investigate native-like functional arrays of the E. coli Asp receptor cytoplasmic fragment (CF), CheA, and CheW assembled on vesicles. Solid-state NMR methods for selective detection of protein interfaces and rigid protein regions will determine structure and structural changes at the receptor/CheA interface. Complementary hydrogen deuterium exchange mass spectrometry (HDX-MS) experiments will determine how CheA domain interactions and linker flexibility change with signaling state.
Aim 3 will first optimize preparations of functional arrays of intact receptors with CheA and CheW, and then apply HDX-MS and selected NMR experiments to this more complex sample to discriminate which signaling-related changes are caused by ligand binding vs receptor methylation (adaptation). These experiments will test our hypothesis that the receptor cytoplasmic domain is partially disordered, and that signaling inputs modulate this disorder to control contacts with CheA and kinase activity. Understanding the mechanism of this key signaling system will yield insights into the roles and mechanisms of disordered domains in other protein complexes and how these contribute to long-range allosteric processes. The project will also demonstrate the promise of combining approaches, such as HDX-MS and solid-state NMR, for advancing mechanistic understanding of the many protein assemblies that play key roles in biology.
This project will measure signaling-related changes in structure and dynamics in native-like functional arrays of chemotaxis receptor complexes, and test the hypothesis that modulating kinase activity involves stabilization of the partially disordered receptor cytoplasmic domain. This will yield insights into mechanisms of long range allosteric coupling and mechanistic roles of disordered protein domains. Methods will be useful for other important protein complexes and mechanistic insights may enable the development of novel antibiotics targeting similar signaling systems that are widespread in bacteria.
