Structural Evidence for Dimer-Interface-Driven Regulation of the Type II Cysteine Desulfurase, SufS

Abstract

SufS is a type II cysteine desulfurase and acts as the initial step in the Suf Fe-S cluster assembly pathway. In Escherichia coli this pathway is utilized under conditions of oxidative stress and is resistant to reactive oxygen species. Mechanistically this means SufS must shift between protecting a covalent persulfide intermediate and making it available for transfer to the next protein partner in the pathway, SufE. Here, we report five x-ray crystal structures of SufS including a new structure of SufS containing an inward facing persulfide intermediate on C364. Additional structures of SufS variants with substitutions at the dimer interface show changes in dimer geometry and suggest a conserved β-hairpin structure plays a role in mediating interactions with SufE. These new structures, along with previous HDX-MS and biochemical data identify an interaction network capable of communication between active-sites of the SufS dimer coordinating the shift between desulfurase and transpersulfurase activities.

Direct observation of intermediates in the SufS cysteine desulfurase reaction reveals functional roles of conserved active-site residues

“…In agreement with previous experiments, this did not yield electron density for a trapped intermediate but instead showed electron density for a sub-stoichiometric amount of Cys-364-persulfide, the product of the cysteine desulfurase reaction (Fig. S4) (23,24). Next, the structure of SufS C364A was solved following incubation of the crystal with L-cysteine.…”

“…The global rotation of the SufS monomers relative to each other in the Cys-ketimine state can be compared with previous studies showing that formation of an active-site persulfide alters SufS dynamics (25). HDX-MS and structural data of SufS dimer interface mutants identified a pathway for active-site cross-talk in the SufS homodimer (23). The conformational changes observed in Fig.…”

Snapshots of PLP‐substrate and PLP‐product external aldimines as intermediates in two types of cysteine desulfurase enzymes

“…8B). Notably, the rotation has recently also been suggested by the results of X-ray structural and biochemical analyses of E. coli SufS [38], which is consistent with the structure-based catalytic mechanism proposed in the present study. The slight change of the loop of B. subtilis SufS is also relevant to a less conformational change of Cys361-SSH of SufS in the sulfurtransferring intermediate of (SufS) 2 -(SufU) 2 than Cys41 of SufU [19].…”

A conserved asparagine in a ubiquitin‐conjugating enzyme positions the substrate for nucleophilic attack

“…, the authors used point mutations in TIM and alkaline phosphatase to shift the rate‐limiting steps such that the covalent intermediates were stuck, allowing them to be crystallized. Similarly, a persulfide intermediate in a mutant SufS system was also trapped and crystallized …”