Crucial Role of an Additional α1-α3 Salt Bridge in Stabilizing the Active Site of Sphingomonas sp. Glutaredoxin 3 for Cold Adaptation.

Abstract

Bacterial glutaredoxin 3 (Grx3) proteins are class I oxidoreductases with a canonical thioredoxin fold. They maintain a conserved glutathione (GSH) interaction site across a range of temperatures, yet their cold adaptation mechanisms remain largely unexplored. In mesophilic Escherichia coli Grx3 (EcGrx3), two conserved α3-helix salt bridges are present, whereas psychrophilic Sphingomonas sp. Grx3 (SpGrx3) features an additional α1-α3 salt bridge (Arg17-Asp68) that is absent in EcGrx3, where Tyr69 occupies the equivalent position. This study investigates how SpGrx3 stabilizes its active site during cold adaptation, focusing on α3-helix salt bridges and aromatic residues. We show that disrupting the C-terminal salt bridge (Lys25 with Glu74-Asp80, between α1-α3) reduces thermal and thermodynamic stability, while disrupting the N-terminal salt bridges (Arg17-Asp68 between α1-α3 and Arg51-Asp69 between α2-α3) diminishes GSH affinity. Substituting α3-helix aromatic residues in SpGrx3 (S67F, S67Y, and A71Y) to mimic the EcGrx3 configuration improves both thermal stability and GSH affinity, whereas the Y69A mutation in EcGrx3-a reciprocal substitution of A71Y in SpGrx3-reduces these properties. These results indicate that Tyr69 is critical for active-site stability in EcGrx3, while its absence in SpGrx3 leads to increased flexibility and reduced GSH affinity, which is partially compensated by the formation of an additional α1-α3 salt bridge during cold adaptation. This study highlights the essential role of α1-α3 helix interactions in preserving the oxidoreductase function of Grx3 proteins across varying temperatures.