Aspartate receptor of bacterial chemotaxis: Structure and on-off switching of the conserved cytoplasmic domain

Bacterial chemotaxis is one of the best characterized two component signaling pathways in which a transmembrane receptor controls a cytoplasmic phosphorelay system, ultimately resulting in the directed movement of the cell toward a favorable chemical environment. The chemoreceptor is responsible for receiving external cues and transmitting them across the bilayer to regulate the histidine kinase CheA.;The thesis focuses on the cytoplasmic domain of the Salmonella typhimurium transmembrane aspartate chemoreceptor Tar. The receptor cytoplasmic domain can be divided into two modules: (i) the cytoplasmic HAMP (domain present in histidine kinases, a denylyl cyclases, methyl-accepting proteins and phosphatase) domain which serves as a signal conversion module, and (ii) the cytoplasmic kinase control module consisting of an adaptation, a coupling, and a protein interaction region that binds the CheA kinase. Both of these modules are widely conserved in bacterial chemoreceptors and must undergo on-off switching transitions during receptor signaling.;The HAMP domain is an essential signal transduction element that interconverts different types of conformational signals between the transmembrane signaling and kinase control modules. A NMR structure was determined for a HAMP domain isolated from an unusual archeal membrane protein. The first study presented herein employed cysteine and disulfide chemistry to test the NMR model for HAMP structure in the full-length, membrane-bound aspartate receptor. The findings show that the NMR HAMP structure is indeed a good model for the HAMP domain of the full-length, membrane-bound aspartate receptor. The findings confirm that the HAMP domain is a parallel 4-helix bundle, and also reveal signal locking disulfide bonds that provide initial insights into the HAMP on-off switching mechanism.;Following this study, the HAMP signal transduction mechanism was investigated using three independent chemical approaches to detect conformational changes triggered by HAMP on-off switching in the full-length, membrane-bound aspartate receptor. Overall, the findings reveal the HD1-HD2' interface is a key on-off switching element where the input piston transmembrane signal is converted into a helix tilt within HAMP. Furthermore, the resulting piston-triggered tilt leads to a working model for the HAMP output signal. The final study develops a new method termed "socket engineering" to carry out a "knob truncation scan" to probe the helix-helix packing interactions in the cytoplasmic kinase control module, which is a long 4-helix bundle.;The findings reveal that signal transmission through this bundle occurs via a novel "Yin-Yang" mechanism. Specifically, strong helix packing in the adaptation region and weak helix packing in the protein interaction region stabilizes on state of the receptor. By contrast, weak helix packing in the adaptation region and strong helix packing in the protein interaction region stabilizes the off state. Thus, the yin-yang on-off switching mechanism generates concerted, antisymmetric helix-helix packing changes within the adaptation and protein interaction regions during receptor on-off switching.;These findings for the cytoplasmic domain of the aspartate chemoreceptor are likely to have broad implications for the large superfamily of bacterial chemoreceptors, which share conserved cytoplasmic HAMP domains, adaptation regions, and protein interaction regions and serve to regulate ubiquitous two-component chemosensory pathways. Moreover, elements of the cytoplasmic domain are found in receptor kinases that control many other types of two-component pathways. Thus, the present findings may well have relevance for most bacterial signaling pathways.