Interaction analysis of TcrX/Y two component system from Mycobacterium tuberculosis.
TcrX/Y is one of the twelve two component system (TCS) present in Mycobacterium tuberculosis. We have investigated the TcrX/Y interaction by in silico studies, pull down assay, radioactive phosphotransfer, surface plasmon resonance as well as crosstalk analysis of TcrY with TcrA - a non-cognate response regulator. Sequence alignment of TcrY with other histidine kinases revealed His256 as the residue responsible for autophosphorylation. The modeled structure of TcrX/Y was docked with each other by GRAMM-X revealing the interaction of TcrY/His256 with TcrX/Asp54. TcrY dimerization via the formation of four helix bundle was also observed by protein-protein docking. Autophosphorylation of TcrY has been observed followed by the phosphate transfer from TcrY to TcrX. The phosphorylation process required divalent metal ions like Mg(2+) or Ca(2+) ions as evident from the radioactive phosphorylation studies. Interaction was not observed between TcrY and TcrA suggesting the signal transduction process is specific in TcrX/Y system. TcrY hydrolyzes ATP and the K(m) value has been found to be 10 mM which is comparable to that of Hsp104. TcrX/Y interaction has been determined by surface plasmon resonance and dissociation constant (K(D)) was evaluated to be 3.6 microM. We conclude from our results that TcrX and TcrY are part of the same signal transduction pathway without their involvement in crosstalk with non-cognate counterpart.
Bhattacharya
Monolekha
M
Department of Biotechnology, Indian Institute of Technology-Kharagpur, Kharagpur, India.
Biswas
Ashis
A
Das
Amit Kumar
AK
D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D002413:Cations, Divalent / Q000378:metabolism; D003432:Cross-Linking Reagents / Q000378:metabolism; D005976:Glutaral / Q000378:metabolism; D000071677:Histidine Kinase; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009169:Mycobacterium tuberculosis / Q000201:enzymology*; D011494:Protein Kinases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D055503:Protein Multimerization; D017433:Protein Structure, Secondary / Q000235:genetics; D017434:Protein Structure, Tertiary / Q000235:genetics; D016415:Sequence Alignment; D015398:Signal Transduction / Q000502:physiology*; D020349:Surface Plasmon Resonance
D001426:Bacterial Proteins; D002413:Cations, Divalent; D003432:Cross-Linking Reagents; D011494:Protein Kinases; D000071677:Histidine Kinase; D005976:Glutaral