Expression and molecular characterization of the Mycobacterium tuberculosis PII protein.
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.
Bandyopadhyay
Anannya
A
Functional Genomics Unit, Institute of Genomics and Integrative Biology (CSIR), Delhi 110 007, India.
Arora
Amit
A
Jain
Sriyans
S
Laskar
Aparna
A
Mandal
Chhabinath
C
Ivanisenko
Vladimir A
VA
Fomin
Eduard S
ES
Pintus
Sergey S
SS
Kolchanov
Nikolai A
NA
Maiti
Souvik
S
Ramachandran
Srinivasan
S
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
D001426:Bacterial Proteins; D007656:Ketoglutaric Acids; D051922:PII Nitrogen Regulatory Proteins; D000244:Adenosine Diphosphate; D000255:Adenosine Triphosphate