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Journal of Biochemistry Advance Access published online on November 2, 2009
Journal of Biochemistry, doi:10.1093/jb/mvp174
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© The authors 2009. Published by Oxford University Press on behalf of the Japanese Biochemical Society. All rights reserved.

Expression and molecular characterization of the Mycobacterium tuberculosis PII protein

Anannya Bandyopadhyay1, Amit Arora2, Sriyans Jain1, Aparna Laskar3, Chhabinath Mandal3, Vladimir A. Ivanisenko4, Eduard S. Fomin4, Sergey S. Pintus4, Nikolai A. Kolchanov4, Souvik Maiti2 and Srinivasan Ramachandran1,*

1 Functional Genomics Unit, Institute of Genomics and Integrative Biology, Delhi 110 007, India. 2 Proteomics and Structural Biology Unit, Institute of Genomics and Integrative Biology, Delhi 110 007, India. 3 Structural Biology & Bioinformatics Division, Indian Institute of Chemical Biology, 4 Raja S.C. Mullick Road, Kolkata 700 032, India. 4 Laboratory of Theoretical Genetics, Institute of Cytology and Genetics, Lavrentieva 10, Novosibirsk 630090, Russia.

* Corresponding Author: Dr. S. Ramachandran, Institute of Genomics and Integrative Biology, Near Jubilee Hall, Mall Road, Delhi 110 007, India, Tel: 0091-11-2766-6156, x169; Fax: 0091-11-2766-7471; Email: ramuigib{at}gmail.com; ramu{at}igib.res.in

Received May 28, 2009; Accepted October 6, 2009


  Abstract

The signal transduction protein PII plays an important role in cellular nitrogen assimilation and regulation. The molecular characteristics of the M. tuberculosis PII (Mtb PII) were investigated using biophysical experiments. The Mtb PII coding ORF Rv2919c was cloned and expressed in Escherichia coli. The binding characteristics of the purified protein with ATP and ADP were investigated using Surface Plasmon Resonance (SPR) and Isothermal Titration Calorimetry (ITC). Mtb PII binds to ATP strongly with Kd in the range 1.93-6.44 µM. This binding strength was not significantly affected by the presence of 2-ketoglutarate even in molar concentrations of 66 (ITC) or 636 (SPR) fold excess of protein concentration. However, an additional enthalpy of 0.3 kcal/mol was released in presence of 2-ketoglutarate. Binding of Mtb PII to ADP was weaker by an order of magnitude. Binding of ATP and 2-ketoglutarate were analysed by docking studies on the Mtb PII crystal structure (PDB id 3BZQ [PDB] ). We observed that hydrogen bonds involving the {gamma}-phosphate of ATP contribute to enhanced binding of ATP compared with ADP. Glutaraldehyde Crosslinking showed that Mtb PII exists in homotrimeric state which is consistent with other PII proteins. Phylogenetic analysis showed that Mtb PII consistently grouped with other actinobacterial PII proteins.


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