Structure and Dynamics of the CheY Response Regulators from Rhodobacter sphaeroides

Abstract

The chemotaxis signalling network of E. coli. depends on autophosphorylation of a histidine protein kinase (HPK) in response to a signal from a sensor domain, with subsequent transfer of the phosphoryl group to an aspartate on response regulator (RR) proteins that bind to the flagellar motor and alter its rotation. CheY is a 14kDa single domain RR that is conserved across motile species. It is formed by 5 α-helices and 5 β-strands surrounding a conserved phosphoryl accepting aspartate residue, and once phosphorylated diffuses to the flagellar motor, binding to its FliM component to cause switching of rotational direction. The photosynthetic bacterium Rhodobacter sphaeroides has multiple chemosensory pathways formed by homologues of the E. coli chemosensory proteins. It has six CheY homologues with different effects on chemotaxis. Only CheY6 is able to stop the flagellar motor but either CheY3 or CheY4 are also required for chemotaxis. NMR and computational methods have been used to answer questions about the structure, dynamics and function of two of the CheY's, CheY3 and CheY6. NOEs, chemical shifts and residual dipolar couplings are used to define the structures of CheY3 and CheY6 in their inactive and active states, where phosphorylation is mimicked using BeF3-. We have investigated fast timescale backbone dynamics using the {1H}-15N heteronuclear NOE and have used CPMG relaxation dispersion experiments to detect low populations of alternative conformations. CheY6 differs from the other R. sphaeroides CheYs and E. coli CheY by the insertion of a ten-residue loop before the C-terminal helix. We have deleted this loop region from CheY6 in order to determine, using in vivo and in vitro assays, if it plays a role in the unique function of CheY6 in R. sphaeroides.