title: Probing the amino acids critical for protein oligomerisation and protein-nucleotide interaction in Mycobacterium tuberculosis PII protein through integration of computational and experimental approaches.
abstract: We investigated the interacting amino acids critical for the stability and ATP binding of Mycobacterium tuberculosis PII protein through a series of site specific mutagenesis experiments. We assessed the effect of mutants using glutaraldehyde crosslinking and size exclusion chromatography and isothermal titration calorimetry. Mutations in the amino acid pair R60-E62 affecting central electrostatic interaction resulted in insoluble proteins. Multiple sequence alignment of PII orthologs displayed a conserved pattern of charged residues at these positions. Mutation of amino acid D97 to a neutral residue was tolerated whereas positive charge was not acceptable. Mutation of R107 alone had no effect on trimer formation. However, the combination of neutral residues both at positions 97 and 107 was not acceptable even with the pair at 60-62 intact. Reversal of charge polarity could partially restore the interaction. The residues including K90, R101 and R103 with potential to form H-bonds to ATP are conserved throughout across numerous orthologs of PII but when mutated to Alanine, they did not show significant differences in the total free energy change of the interaction as examined through isothermal titration calorimetry. The ATP binding pattern showed anti-cooperativity using three-site binding model. We observed compensatory effect in enthalpy and entropy changes and these may represent structural adjustments to accommodate ATP in the cavity even in absence of some interactions to perform the requisite function. In this respect these small differences between the PII orthologs may have evolved to suite species specific physiological niches.
authors.affiliation: CSIR-Institute of Genomics and Integrative Biology, Mall Road, New Delhi 110 007, India.
authors.forename: Sriyans
authors.initials: S
authors.lastname: Jain
authors.forename: Rahul Shubhra
authors.initials: RS
authors.lastname: Mandal
authors.forename: Swadha
authors.initials: S
authors.lastname: Anand
authors.forename: Souvik
authors.initials: S
authors.lastname: Maiti
authors.forename: Srinivasan
authors.initials: S
authors.lastname: Ramachandran
mesh_terms: D000255:Adenosine Triphosphate / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D019943:Amino Acid Substitution; D000596:Amino Acids; D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D019143:Evolution, Molecular; D008958:Models, Molecular; D020125:Mutation, Missense; D009169:Mycobacterium tuberculosis / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D051922:PII Nitrogen Regulatory Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D011485:Protein Binding; D055503:Protein Multimerization / Q000502:physiology*; D020836:Protein Structure, Quaternary; D017434:Protein Structure, Tertiary
chemical_list: D000596:Amino Acids; D001426:Bacterial Proteins; D051922:PII Nitrogen Regulatory Proteins; C404143:PIID regulatory protein, Bacteria; D000255:Adenosine Triphosphate
keywords: 2-OG; 2-oxoglutarate; AMPPNP; ATP-γS; Adenosine 5′-(β,γ-imido) triphosphate; Adenosine 5′-[γ-thio]-triphosphate; Electrostatic interactions; ITC; Isothermal Titration Calorimetry; Isothermal titration calorimetry; Molecular docking; MtbPII; Mutagenesis; Mycobacterium tuberculosis PII protein; PIC; PISA; Protein Interaction Calculator server; Protein Interfaces, Surfaces and Assemblies server; Protein stability