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Interaction analysis of TcrX/Y two component system from Mycobacterium tuberculosis
Monolekha Bhattacharya1, Ashis Biswas, Amit Kumar Das
1Department of Biotechnology, Indian Institute of Technology-Kharagpur, Kharagpur, India.
Biochimie
|December 8, 2009
Summary
The TcrX/Y two-component system in Mycobacterium tuberculosis is specific, with TcrY autophosphorylating and transferring phosphate to TcrX. This signal transduction pathway avoids crosstalk with non-cognate regulators.
Area of Science:
- Microbiology
- Molecular Biology
- Biochemistry
Background:
- Mycobacterium tuberculosis possesses twelve two-component systems (TCS).
- The TcrX/Y system is one such TCS requiring investigation into its specific interactions and function.
Purpose of the Study:
- To elucidate the interaction mechanism between TcrX and TcrY in Mycobacterium tuberculosis.
- To determine the specificity of the TcrX/Y signal transduction pathway.
Main Methods:
- In silico studies including protein-protein docking (GRAMM-X).
- Biochemical assays: pull-down assay, radioactive phosphotransfer, and surface plasmon resonance (SPR).
- Kinetic analysis of ATP hydrolysis and metal ion dependency.
Main Results:
- His256 in TcrY was identified as the autophosphorylation residue, interacting with Asp54 in TcrX.
- TcrY dimerization and autophosphorylation were observed, followed by phosphate transfer to TcrX, requiring Mg(2+) or Ca(2+).
- SPR analysis yielded a dissociation constant (KD) of 3.6 μM for TcrX/Y interaction, and TcrY exhibited ATP hydrolysis with a Km of 10 mM.
Conclusions:
- TcrX and TcrY function within the same specific signal transduction pathway.
- The TcrX/Y system demonstrates no crosstalk with the non-cognate regulator TcrA, confirming pathway specificity.
