Investigation of protein-protein interactions in the suf pathway for fe-s cluster assembly in escherichia coli
Fe-S cluster cofactor biogenesis is critical to the survival of bacterial pathogens and is carried out by multiprotein biosynthetic pathways. In E. coli, the SUF system is one of three distinct pathways for Fe-S cluster biosynthesis and is utilized under stress conditions. SufS is a group II cysteine desulfurase in the SUF system and requires its partner protein, SufE to mobilize persulfide from L-cysteine. To better understand the molecular details of how SufS and SufE interact, we applied backbone amide hydrogen-deuterium exchange mass spectrometry (HDX-MS) combined with biochemical and biophysical assays. We report results of HDX-MS experiments aimed at identifying changes in the protein dynamics of SufS in complex with the activated variants of SufE and upon persulfuration of SufS. HDX-MS analysis of SufED74R revealed an increase in solvent accessibility and dynamics in the loop containing the active site Cys51 used to accept persulfide from SufS. Importantly, ITC studies show that SufEapo binds to SufSapo in a two-site model with negative cooperativity. However, the modified SufED74R binds with a one-site model and a 10-fold increase in affinity. Furthermore, SufED74R exhibits a stoichiometry of 0.57 suggesting that it only binds to one monomer of SufS at a time. These results point toward an additional level of regulation through a half-sites mechanism that affects the stoichiometry and affinity for SufE as the dimeric SufS shifts between desulfurase and transpersulfuration activities. Investigation of the covalent persulfide intermediate of SufS by HDX-MS identified two active site peptides and two peptides at the dimer interface of SufS that exhibit changes in deuterium uptake upon formation of the intermediate. Residues in these peptides are organized to form a conduit between the two active sites upon persulfide formation and include key cross-monomer interactions. Three evolutionarily conserved residues at the dimer interface were investigated by alanine scanning mutagenesis. Two variants resulted in 6-fold increases in the value of KSufE, confirming a functional role. The identification of conformational changes at the dimer interface and structural reports provides a physical mechanism for active site communication in the half-sites regulation of SufS activity. Given the conservation of the interface interactions, this mechanism may be broadly applicable to group II cysteine desulfurase systems.