Structures of the ON and OFF states of the membraneous fumarate/Citrate sensor kinases DcuS from E. coli and CitA from Geobacillus thermodenitrifcans

• Applicants: Professor Dr. Christian Griesinger; Professor Dr. Dariush Hinderberger; Professor Dr. Gottfried Unden
• Subject Area: Metabolism, Biochemistry and Genetics of Microorganisms
• Term: from 2017 to 2021
• Project identifier: Deutsche Forschungsgemeinschaft (DFG) - Project number 396465415

Sensor kinases (SK) represent a major device of bacteria for responding to extracellular stimuli, resulting in autophosphorylation of the SK. Their function includes signal perception, transmembrane (TM) signaling, and signal transmission to the kinase. Domain composition and transmembrane signaling of the SK show broad variation. The proposal aims at solving the structure of a SK of the DcuS/CitA family (fumarate and citrate sensing) in the ON and the OFF state, and to understand restructuring of the domains in transmembrane and intracellular signaling. DcuS/CitA contain an archaetypical domain composition for transmembrane signaling SK. From the vast number of TM sensor kinases, the structure of the NarQ nitrate SK has been solved in a form depleted of the cytosolic GAF and kinase domains recently. NarQ represents a unique SK with a domain composition largely different from DcuS/CitA and stands very likely for a different mode of signal transduction. DcuS of E. coli and the closely related CitA of the thermophilic Geobacillus thermodenitrificans are homodimers each, composed of an extracytoplasmic PASP (Per ARNT Sim) domain for fumarate or citrate binding, two TM helices (TM1, TM2), a cytoplasmic PASC and the kinase domain. Studies are performed with DcuS or CitA complementarily according to availability of the proteins for the analytic systems. Structures of PASP and PASC have been solved in isolated and membrane-integral constructs in the ON and OFF states. Structural, genetic and biochemical studies show a compaction of PASP upon substrate binding that triggers a piston-like movement of TM2 which is responsible for transmembrane signaling. PASC responds to the shift of TM2 in a structural rearrangement at an alpha-helical dimerization site. This work is the basis for high resolution analysis and an integrated view of the structural rearrangements in the domains during transmembrane signaling. Work will concentrate on (i) on the structural rearrangements in the TM region with the piston-like movement of TM2 that may be combined with a scissors movement of TM2, (ii) the structural rearrangement in PASc during signal transmission and control of the kinase domain. For a complete view and high resolution, structural (NMR and crystallization; AG Griesinger), biochemical, genetic (AG Unden) and biophysical (AG Hinderberger, Griesinger) methods will be applied. Preparatory work established all constructs and the methods for the study, and we presume that DcuS/CitA represent one of the very few SK whose transmembrane and cytoplasmic signaling is within reach at high structural and functional resolution by a combined approach from structural biology, biochemistry and biophysics.

• DFG Programme: Research Grants