Probing the amino acids critical for protein oligomerisation and protein–nucleotide interaction in Mycobacterium tuberculosis PII protein through integration of computational and experimental approaches

• Published in: Biochimica et biophysica acta 2013-12, Vol.1834 (12), p.2736-2749
• Main Authors: Jain, Sriyans, Mandal, Rahul Shubhra, Anand, Swadha, Maiti, Souvik, Ramachandran, Srinivasan
• Format: Article
• Language: English
• Subjects: adenosine triphosphate; Adenosine Triphosphate - chemistry; Adenosine Triphosphate - genetics; Adenosine Triphosphate - metabolism; alanine; Amino Acid Substitution; Amino Acids; Bacterial Proteins - chemistry; Bacterial Proteins - genetics; Bacterial Proteins - metabolism; calorimetry; crosslinking; Electrostatic interactions; energy; enthalpy; entropy; Evolution, Molecular; gel chromatography; glutaraldehyde; Isothermal titration calorimetry; Models, Molecular; Molecular docking; Mutagenesis; mutants; Mutation, Missense; Mycobacterium tuberculosis; Mycobacterium tuberculosis - chemistry; Mycobacterium tuberculosis - genetics; Mycobacterium tuberculosis - metabolism; niches; PII Nitrogen Regulatory Proteins - chemistry; PII Nitrogen Regulatory Proteins - genetics; PII Nitrogen Regulatory Proteins - metabolism; Protein Binding; Protein Multimerization - physiology; Protein stability; Protein Structure, Quaternary; Protein Structure, Tertiary; proteins; sequence alignment; titration
• ISSN: 1570-9639; 0006-3002; 1878-1454
• DOI: 10.1016/j.bbapap.2013.10.006

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. •Amino acid pair R60-E62 form critical interaction to stabilise MtbPII trimer.•Surface located amino acid pairs play supportive role in stability of MtbPII trimer.•Charge reversal of interacting residues only partially restores the interaction.•ATP binding with MtbPII shows anti-cooperativity.•ATP binding is preserved through enthalpy–entropy compensation in MtbPII mutants.