Expression, purification, and characterization of the histidine kinase CarS from Fusobacterium nucleatum / 生物工程学报

Fusobacterium nucleatum is an opportunistic pathogenic bacterium that can be enriched in colorectal cancer tissues, affecting multiple stages of colorectal cancer development. The two-component system plays an important role in the regulation and expression of genes related to pathogenic resistance and pathogenicity. In this paper, we focused on the CarRS two-component system of F. nucleatum, and the histidine kinase protein CarS was recombinantly expressed and characterized. Several online software such as SMART, CCTOP and AlphaFold2 were used to predict the secondary and tertiary structure of the CarS protein. The results showed that CarS is a membrane protein with two transmembrane helices and contains 9 α-helices and 12 β-folds. CarS protein is composed of two domains, one is the N-terminal transmembrane domain (amino acids 1-170), the other is the C-terminal intracellular domain. The latter is composed of a signal receiving domain (histidine kinases, adenylyl cyclases, methyl-accepting proteins, prokaryotic signaling proteins, HAMP), a phosphate receptor domain (histidine kinase domain, HisKA), and a histidine kinase catalytic domain (histidine kinase-like ATPase catalytic domain, HATPase_c). Since the full-length CarS protein could not be expressed in host cells, a fusion expression vector pET-28a(+)-MBP-TEV-CarScyto was constructed based on the characteristics of secondary and tertiary structures, and overexpressed in Escherichia coli BL21-Codonplus(DE3)RIL. CarScyto-MBP protein was purified by affinity chromatography, ion-exchange chromatography, and gel filtration chromatography with a final concentration of 20 mg/ml. CarScyto-MBP protein showed both protein kinase and phosphotransferase activities, and the MBP tag had no effect on the function of CarScyto protein. The above results provide a basis for in-depth analysis of the biological function of the CarRS two-component system in F. nucleatum.

Progress in the understanding of the function of atypical response regulators: a review / 生物工程学报

Two component system is a signal transduction system. It typically consists of a sensor histitine kinase and a cognate response regulator (RR) component. The activity of RR is regulated by a phosphorylation dependent mechanism. In recent years, the existence of atypical response regulators (ARRs), which rely on a phosphorylation independent mechanism to regulate their activity, have been recognized. ARRs are involved in the regulation of bacterial growth and development, antibiotic biosynthesis, iron transport, among others. Here we review the recent advances in the understanding of the structure and function of atypical response regulators, by using JadR1, a regulator in jadomycin biosynthesis in Streptomyces, as an example to elucidate the novel mechanism used by ARR to fine-tune its activity.

Histidine kinase (YycG) protein of Streptococcus pneumoniae: homology modeling and analysis / 生物工程学报

To construct a three-dimensional (3D) model of histidine kinase (HK) YycG protein in Streptococcus pneumoniae and to investigate the interaction between YycG and its substrate ADP for the purpose of providing a theoretical basis for YycG selective inhibitor discovery, we constructed a 3D model of YycG protein by homology modeling, and assessed the reliability of the model using ProCheck and Profile_3D software. Besides, the active-site cavity of YycG and the residues key for substrate interaction were analyzed by Autodock4.0. Sequence alignment indicated that the YycG of S. pneumoniae was homologous to that of Thermotoga maritima. The constructed 3D model of YycG adopted a similar folding pattern to the template and the two matched well. The conservative amino acids in the substrate-binding pocket, such as Asn145, Asn149 and Lys152, as well as the hydrophobic residues at the bottom of the pocket played important role in binding and hydrolyzing substrate ADP. We have successfully constructed a reliable model of YycG protein. The model can be used as a starting point for designing antibacterial drugs.