title: Expression and molecular characterization of the Mycobacterium tuberculosis PII protein.
abstract: The signal transduction protein PII plays an important role in cellular nitrogen assimilation and regulation. The molecular characteristics of the Mycobacterium 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 K(d) in the range 1.93-6.44 microM. 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). 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.
authors.affiliation: Functional Genomics Unit, Institute of Genomics and Integrative Biology (CSIR), Delhi 110 007, India.
authors.forename: Anannya
authors.initials: A
authors.lastname: Bandyopadhyay
authors.forename: Amit
authors.initials: A
authors.lastname: Arora
authors.forename: Sriyans
authors.initials: S
authors.lastname: Jain
authors.forename: Aparna
authors.initials: A
authors.lastname: Laskar
authors.forename: Chhabinath
authors.initials: C
authors.lastname: Mandal
authors.forename: Vladimir A
authors.initials: VA
authors.lastname: Ivanisenko
authors.forename: Eduard S
authors.initials: ES
authors.lastname: Fomin
authors.forename: Sergey S
authors.initials: SS
authors.lastname: Pintus
authors.forename: Nikolai A
authors.initials: NA
authors.lastname: Kolchanov
authors.forename: Souvik
authors.initials: S
authors.lastname: Maiti
authors.forename: Srinivasan
authors.initials: S
authors.lastname: Ramachandran
mesh_terms: D000244:Adenosine Diphosphate / Q000378:metabolism; D000255:Adenosine Triphosphate / Q000378:metabolism; D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism*; D002151:Calorimetry; D002942:Circular Dichroism; D006860:Hydrogen Bonding; D007656:Ketoglutaric Acids / Q000378:metabolism; D009169:Mycobacterium tuberculosis / Q000145:classification / Q000235:genetics / Q000378:metabolism*; D051922:PII Nitrogen Regulatory Proteins / Q000235:genetics / Q000378:metabolism*; D010802:Phylogeny; D011485:Protein Binding; D020349:Surface Plasmon Resonance
chemical_list: D001426:Bacterial Proteins; D007656:Ketoglutaric Acids; D051922:PII Nitrogen Regulatory Proteins; D000244:Adenosine Diphosphate; D000255:Adenosine Triphosphate