Functional significance of highly conserved residues in dithiol glutaredoxin (Grx3) of cyanobacterium Synechococcus elongatus PCC 7942.

Glutaredoxins regulate cellular redox homeostasis, protect against oxidative stress, and modulate protein function crucial for the fitness of photosynthetic organisms and diseases progression in human. Protein functionality is intricately linked to structural integrity, which is maintained by conserved amino acids residues. This study investigates the structural and functional effect of single-point mutations at evolutionarily highly conserved residues in the dithiol glutaredoxin (Grx3) protein of cyanobacterium Synechococcus elongatus PCC 7942. Conservation analysis identified key residues such as catalytic cysteines (Cys12, Cys15), a cis-proline (Pro55), and glycine residues within the GG-kink motif (Gly65, Gly66) as important determinants of enzymatic stability and activity. Mutational analyses and molecular dynamics simulations revealed that substitutions at Cys15 and the GG kink significantly compromise thermodynamic stability, structural compactness, and hydrogen bonding networks. However, Pro55 and Cys12 substitutions showed context-dependent effects on protein flexibility and solvent exposure. Notably, C15A demonstrated enhanced structural rigidity and compactness, supported by stable hydrogen bonding and favourable radius of gyration metrics. Conversely, mutations like G65A and G66A, on the other hand, caused significant structural changes and lowered binding affinities with glutathione due to disruption of key hydrophobic and hydrogen bond interactions. Binding free energy and molecular dynamics simulations further elucidated the allosteric consequences of local mutations, which also highlighted how they affect global conformational dynamics and substrate accommodation. These results highlight the necessity of conserved residues for structure and function and provide a framework for structure-guided engineering of redox-active proteins for biotechnological applications.