Molecular mechanisms of signal transduction in two-component signaling systems
Year: 2013
Type: dissertation
Abstract: Two-component signaling systems is an important class of proteins. In contrast to eukaryota, most prokaryotic signal transduction pathways use schemes of phosphate transfer involving two conserved components, a histidine protein kinase and a response regulator protein. To the moment, hundreds of such systems were found in eubacteria, archaea, and a few eukaryotic organisms. These systems serve as a basic stimulus-response coupling mechanism to allow organisms to sense and respond to changes in many different environmental conditions. Though many advances in the field of signal transduction in two-component signal transduction have been made recently, there is still a strong demand for robust model systems where molecular mechanism of signal transduction can be studied. The complex of two membrane proteins from Halobacterium N.pharaonis, Sensory Rhodopsin II (SRII) with its cognate Transducer (HtrII) is an example of such system. It is serving as a phototaxis receptor that helps halobacteria to avoid harmful UV light. Sensory rhodopsin II belongs to the family of archaeal retinal proteins. HtrII is highly homologous to bacterial chemoreceptors. It consists of transmembrane domain, linker domain, methylation region involved in adaptation and kinase domain. The X-Ray structures of SRII/HtrII give a unique insight into the complex of two transmembrane proteins. These structures elucidate initial steps of signal transduction in SRII and possible mechanism to transfer the signal to HtrII. NMR structures of cytoplasmic domain of osmotic sensor EnvZ from E.coli have shown the design of kinase domain that is highly conserved among all histidine kinases. Despite some structural information available there is still no detailed molecular mechanism of functioning of the complex from the initial step of retinal isomerization to phosphate group transition to the kinase CheA. This work is aimed to elucidate some of these steps in more detail and to show by means of X-Ray structural analysis the key features of protein structures that are prerequisite for efficient signal transduction. All halobacterial retinal proteins share common features of the signal transduction mechanism. One of these features is a proton transfer from the Schiff base to the counterion after retinal isomerization. Various studies have shown that the mutation of the counterion to a neutral amino acid residue is critical for the function of most of them. In the case of SRII/HtrII complex, physiological experiments have shown that Asp75-to-Asn75 mutation doesn’t inhibit the signal transduction that is seen as persistent rate of cell motion reversals upon light illumination. This fact is important for understanding signal transduction mechanism in these systems. The structure described in this thesis shows the change in the hydrogen network of SRII-D75N/HtrII complex with respect to the wild type complex. Comparison with the ground and M states of the wild type has helped to elucidate how the signal transduction process might evolve in the D75N mutant protein. Intense crystallization under various conditions has resulted into another finding that might be very important for understanding of the mechanism of functioning. Crystals of different space groups were produced and the structures in these space groups were compared to the previously published structures in the P21212 space group. A striking difference in the arrangement of the SRII and HtrII proteins in the complex were observed. Whereas in the P21212 space group the proteins form a crevice with an opening towards cytoplasmic side (“V”-shape), in newly obtained structure of I212121 space group proteins are roughly parallel to each other (“U”-shape). This thesis contains a discussion about which of the quaternary structures of the complex represents the functional complex that occurs in vivo. Histidine kinase chemoreceptors are another representative of two-component signal transduction systems. NarQ and NarX belong to the family of histidine kinase receptors and react to nitrate or nitrite to regulate anaerobic respiration in various bacteria. This study has revealed some approaches that can be used in studying these very important proteins. First crystals were obtained. The membrane domain of the nitrate/nitrite receptor NarQ from E.coli was successfully expressed, purified and crystallized. At the moment these crystals require additional optimization.
Source: RWTH Publications (RWTH Aachen)
Author: Ishchenko, Andrii
Language: English
Subfield: Genetics
Domain: Life Sciences
Open Access status: green
OpenAlex ID: W637732001