Transmembrane Signalling: Structural and Functional Studies on Histidine Kinase CitA

by Benjamin Schomburg
Doctoral thesis
Date of Examination:2015-01-28
Date of issue:2015-12-03
Advisor:Prof. Dr. Christian Griesinger
Referee:Prof. Dr. Christian Griesinger
Referee:Prof. Dr. Claudia Steinem
Persistent Address: http://dx.doi.org/10.53846/goediss-5409

 

 

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Abstract

English

Bacteria utilise two component systems (TCS) consisting of a homodimeric receptor histidine kinase (HK) and a response regulator (RR) as a prevalent mechanism of stimulus perception and signal transduction. TCSs are key players in the regulation of metabolism, motility and development, and in addition are crucial for virulence in a number of pathogenic species. The study of TCSs is therefore motivated by their importance as a fundamental and widely used signalling system. In this study, the Geobacillus thermodenitrificans Citrate receptor A (CitA) is used as a model system for HKs. Free citrate is recognised by a periplasmic PAS (Per-Arnt- Sim) domain (PASp) and the input signal is then relayed to a second, cytosolic PAS domain (PASc) before leading to auto-phosphorylation in the conserved kinase core. Even though the phosphate transfer mechanism of the kinase has been described in depth, signal transduction across the membrane remains poorly understood. The aim of this PhD project therefore is to elucidate the signalling mechanism of CitA by means of combining liquid- and solid-state-NMR spectroscopy with X-ray crystallography. Our results show a shortening of the C-terminal beta-strand of CitA PASp by one residue upon citrate binding, potentially exerting a pull on the second transmembrane helix. The restructuring of the C-terminus of PASp is in agreement with previously published results on an isolated citrate-binding PAS domain and a piston model for transmembrane helix motion. Additionally, liposome-embedded CitA constructs for the first time allow monitoring changes in the cytosol upon periplasmic citrate binding. In the citrate-bound state, PASc mobility increases, suggesting a constraining role of PASc keeping the kinase domain in an inactive conformation until CitA is triggered.
Keywords: Histidine Kinase; Solid-State NMR; Transmembrane Signaling
 
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