NMR structural studies on the periplasmic domain of CitA and DcuS.

Abstract

Two-component regulatory system represent the most frequent system for transmembrane signaling in bacteria and play a major role in the cellular adaptation to environmental conditions and stress. They consist of two separate proteins, a sensory histidine protein kinase which is located typically in the membrane, and a cognate response regulator in the cytoplasm. Despite the wealth of molecular biological studies in these systems, no structural informations are available on the signal transduction by these systems across the membrane. The aim of the study was to gain structural and dynamic information on signal perception and signal transduction from the periplasmic sensor domain of the two component membraneous sensor into the cytoplasmic domain. In this thesis NMR structural studies on the periplasmic domain of two histidine kinase are presented. DcuS and CitA are bacterial membraneous sensory histidine kinases. They are part of a two component signal transduction systems that regulate the transport and metabolism of di- and tri-carboxylates in response to their environmental concentration. Their periplasmic domains (DcuS-PD and CitAP), are homologous, share a PAS fold, and contain the binding site for the carboxylates. CitA works as a highly specific citrate receptor whereas DcuS uses a wider range of C4 dicarboxylates like fumarate, succinate etc as stimulus. As a first step to understand the signal transfer process, the NMR solution structure of periplasmic domain of DcuS was determined. The structure was refined with residual dipolar couplings (RDCs), measured using a novel strategy for simultaneous measurement of RDCs with minimum resonance overlap. The binding pocket of DcuS-PD for C4 di-carboxylates was defined using 15N-1H HSQC based titrations. The effect of the ligand binding to DcuS-PD was weak. No chemical shift changes or intensity increase for residues were observed outside the binding pocket and hence the signal transduction mechanism remained undetermined. Therefore the sensory domain of CitAP which binds citrate more specifically was studied to obtain a better understanding of the conformational changes that lead to signal transduction. The NMR solution structures of CitAP could not be determined because of the large number of missing peaks due to severe line broadening observed in the NMR spectra. Conformational exchange was the major cause of line broadening. However the X-ray structures of citrate free and bound form of CitAP could be determined. The major conformational changes were observed in the citrate binding region and in the C-terminal region of the protein. Large chemical shift changes and Het-NOE values were also observed in these parts of the protein. In the citrate bound structure, a Na+ ion was tentatively localized between N terminal helix and the beta-sheets. This was also confirmed by NMR titrations. Hence CitAP may be involved in sensing both citrate and Na+ ion in solution. Surprisingly the RDCs measured for citrate free CitAP fit better with citrate bound structure of CitAP. This indicates a pre-formed binding pocket of CitAP in solution. Nevertheless, the specific structural differences between the citrate free and bound structures allowed to formulate a model for the mechanism of signal transduction. This model is consistent with available NMR data and also very similar to the signal transduction mechanism described for aspartate sensors.