Structural And Biochemical Insights Into The Multiple Functions Of Yeast Grx3

Reactive oxygen species(ROS)can cause oxidative damage to biological macromolecule.Living cells utilize two groups of oxireductases,namely glutaredoxins and thioredoxins to maintain the intracellular redox homeostasis.The yeast Sacchromyces cerevisiae encodes three dithiol glutaredoxins:Grx1,Grx2 and Grx8,in addition to five monothiol glutaredoxins:Grx3,Grx4,Grx5,Grx6 and Grx7 to resist oxidative stress or regulate cell iron metabolism.The grx3grx4 double mutant is highly sensitive to oxidative stress,leading to a growth and cell reproduction defect as a consequence of iron-sensing transcription factor Aft2 upregulation.It indicates that Grx3 or Grx4 plays a key role in cellular defense against oxidative stress and more importantly,cooperates with BolA-like iron repressor of activation protein Fra2 to regulate the localization of Aft2.The cytosolic monothiol glutaredoxins Grx3 and Grx4 are highly homologous isoforms that belong to a subgroup of the Grxs featured with an N-terminal Trx domain followed by a C-terminal Grx domain.The Grx domain plays a role in ROS detoxification and cell viability.Howerver,the crosstalk between the two domains of Grx3/4 and the interplay among Grx3/4,Fra2 and Aft2 responsible for the regulation of iron homeostasis has not been clearly described.Here we solved the crystal structures of the Trx domain and Grx domain of Grx3 in addition to the solution structure of Fra2.Structural analyses and activity assays indicated that the Trx domain also contributes to the glutathione S-transferase activity of Grx3,via an inter-domain disulfide bond between Cys37 and Cysl76.NMR titration and pull-down assays combined with surface plasmon resonance experiments revealed that Fra2 could form a noncovalent heterodimer with Grx3 via an interface between the helix-turn-helix motif of Fra2 and the C-terminal segment of Grx3Grx,different from the previously identified covalent heterodimer mediated by Fe-S cluster.Comparative affinity assays indicated that the interaction between Fra2 and Aft2 is much stronger than that between Grx3 and Aft2,or Aft2 towards its target DNA.These structural and biochemical analyses enabled us to propose a model how Grx3 executes multiple functions to coordinate the regulation of Aft2-controlled iron metabolism.