Structural and thermodynamic characterization of a highly stable conformation of Rv2966c, a 16S rRNA methyltransferase, at low pH

• Published in: International journal of biological macromolecules Vol. 164; pp. 3909 - 3921
• Main Authors: Khan, Sabab Hasan, Bijpuria, Shipra, Maurya, Anjali, Taneja, Bhupesh
• Format: Journal Article
• Language: English
• Published: Netherlands Elsevier B.V 01-12-2020
• Subjects: 16S rRNA methyltransferase; Calorimetry, Differential Scanning; Circular Dichroism; Hydrogen-Ion Concentration; Methyltransferases - chemistry; Models, Molecular; Molecular Conformation; Molecular Dynamics Simulation; Mycobacterium tuberculosis; Protein Conformation; Protein folding; RNA, Ribosomal, 16S - chemistry; Rv2966c; Structure-Activity Relationship; Thermodynamics; Urea - chemistry; Urea-induced denaturation; Urea-induced denaturation; Mycobacterium tuberculosis; Protein folding; Rv2966c; 16S rRNA methyltransferase
• ISSN: 0141-8130; 1879-0003
• DOI: 10.1016/j.ijbiomac.2020.08.236

Rv2966c is a highly specific methyltransferase that methylates G966 at the N2 position in 16S rRNA of mycobacterial ribosome and can be secreted inside the host cell to methylate host DNA. However, how the secreted protein retains its structure and function in the harsh environment of host cell, remains unclear. In this work, we investigate structural features of Rv2966c at pH 4.0 and pH 7.5 by far-UV- and near-UV-circular dichroism (CD) and fluorescence spectroscopy, to gain insights into its folding and stability at the acidic pH, that it is likely to encounter inside the macrophage. We show that Rv2966c exists in a compact, folded state at both pH 7.5 and pH 4.0, a result corroborated by molecular dynamics simulations as a function of pH. In fact, Rv2966c was found to be more stable at pH 4.0 than pH 7.5, as evidenced by thermal-induced CD and nanodifferential scanning fluorimetry, and urea-induced denaturation measurements. Interestingly, unlike pH 7.5 (two-state unfolding), denaturation of Rv2966c at pH 4.0 occurs in a biphasic (N ↔ X ↔ U) manner. Further spectroscopic characterization of ‘X’ state, identifies characteristics of a molten globule-like intermediate. We finally conclude that Rv2966c maintains a compact folded state at pH 4.0 akin to that at pH 7.5 but with higher stability.
•Provides insights into folding and stability of Rv2966c at acidic pH•Rv2966c exists in a compact, folded state at pH 4.0.•With a higher Cm (urea) and higher Tm-app at pH 4.0, is more stable than at pH 7.5.•Urea-induced denaturation at pH 4.0 is a biphasic (N ↔ X ↔ U) process.•‘X’ state found to exhibit characteristics of a molten globule-like intermediate.