Structure and Dynamics of the Receptor:Kinase Complex that Mediates Bacterial Chemotaxis

Brian R. Crane
2015 Biophysical Journal  
CheA and CheW, peptides exhibit protection from exchange at long times (16 hours) that is greater in the kinase-on state. HDX-MS of complexes prepared using different means of shifting the signaling state will reveal which changes correlate with kinase activity and will quantify stabilization of receptor subdomains and binding interfaces that contribute to receptor control of kinase activity. Thus HDX-MS provides an important tool in a hybrid approach for understanding structure and mechanism
more » ... membrane-bound, multi-protein complexes. The core unit of the bacterial chemosensory array is multi-protein complex comprised of 6 transmembrane chemoreceptor homodimers, 1 CheA His kinase homodimer, and 2 CheW adaptor protein monomers. We are reconstituting core units on isolated bacterial membranes, yielding individual core units and oligomers of core units ranging in size up to small hexagonal arrays. This approach generates functional, membrane-bound core complexes and allows incorporation of modified kinase and adaptor proteins possessing pairs of engineered Cys residues for disulfide trapping, or spectroscopic probes for fluorescence or EPR studies. The resulting core complexes display native receptor-stimulated kinase activities that are fully regulated by attractant binding to the receptor, or covalent modification of the receptor adaptation sites. The findings shed new light on the kinetics and order of core complex assembly, and provide insights into the molecular mechanisms underlying receptormediated kinase on-off switching.
doi:10.1016/j.bpj.2014.11.258 fatcat:qwvq2q6zvbh4xmpwc2xre72o44