Dataset Information

Crystal Structure of Bacillus subtilis Cysteine Desulfurase SufS and Its Dynamic Interaction with Frataxin and Scaffold Protein SufU.

ABSTRACT: The biosynthesis of iron sulfur (Fe-S) clusters in Bacillus subtilis is mediated by a SUF-type gene cluster, consisting of the cysteine desulfurase SufS, the scaffold protein SufU, and the putative chaperone complex SufB/SufC/SufD. Here, we present the high-resolution crystal structure of the SufS homodimer in its product-bound state (i.e., in complex with pyrodoxal-5'-phosphate, alanine, Cys361-persulfide). By performing hydrogen/deuterium exchange (H/DX) experiments, we characterized the interaction of SufS with SufU and demonstrate that SufU induces an opening of the active site pocket of SufS. Recent data indicate that frataxin could be involved in Fe-S cluster biosynthesis by facilitating iron incorporation. H/DX experiments show that frataxin indeed interacts with the SufS/SufU complex at the active site. Our findings deepen the current understanding of Fe-S cluster biosynthesis, a complex yet essential process, in the model organism B. subtilis.

SUBMITTER: Blauenburg B

PROVIDER: S-EPMC4934914 | biostudies-literature | 2016

REPOSITORIES: biostudies-literature

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Crystal Structure of Bacillus subtilis Cysteine Desulfurase SufS and Its Dynamic Interaction with Frataxin and Scaffold Protein SufU.

Blauenburg Bastian B Mielcarek Andreas A Altegoer Florian F Fage Christopher D CD Linne Uwe U Bange Gert G Marahiel Mohamed A MA

PloS one 20160706 7

The biosynthesis of iron sulfur (Fe-S) clusters in Bacillus subtilis is mediated by a SUF-type gene cluster, consisting of the cysteine desulfurase SufS, the scaffold protein SufU, and the putative chaperone complex SufB/SufC/SufD. Here, we present the high-resolution crystal structure of the SufS homodimer in its product-bound state (i.e., in complex with pyrodoxal-5'-phosphate, alanine, Cys361-persulfide). By performing hydrogen/deuterium exchange (H/DX) experiments, we characterized the inter ...[more]

PMID: 27382962

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Project description:In the Suf Fe-S cluster assembly pathway, the activity of the cysteine desulfurase, SufS, is regulated by interactions with the accessory sulfotransferase protein, SufE. SufE has been shown to stimulate SufS activity, likely by inducing conformational changes in the SufS active site that promote the desulfurase step and by acting as an efficient persulfide acceptor in the transpersulfuration step. Previous 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 backbone amide hydrogen-deuterium exchange mass spectrometry identified two active site peptides (residues 225-236 and 356-366) and two peptides at the dimer interface of SufS (residues 88-100 and 243-255) 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, suggesting they may play a role in the half-sites regulation. Three evolutionarily conserved residues at the dimer interface (R92, E96, and E250) were investigated by alanine scanning mutagenesis. Two of the substituted enzymes (E96A and E250A SufS) resulted in 6-fold increases in the value of KSufE, confirming a functional role. Re-examination of the dimer interface in reported crystal structures of SufS and the SufS homologue CsdA identified previously unnoticed residue mobility at the dimer interface. The identification of conformational changes at the dimer interface by hydrogen-deuterium exchange confirmed by mutagenesis 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 type II cysteine desulfurase systems.

Dataset Information

Crystal Structure of Bacillus subtilis Cysteine Desulfurase SufS and Its Dynamic Interaction with Frataxin and Scaffold Protein SufU.

Publications

Crystal Structure of Bacillus subtilis Cysteine Desulfurase SufS and Its Dynamic Interaction with Frataxin and Scaffold Protein SufU.

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