Proteomic Analysis of Protein−Protein Interactions within the Cysteine Sulfinate Desulfinase Fe−S Cluster Biogenesis System

Abstract

Fe-S cluster biogenesis is of interest to many fields, including bioenergetics and gene regulation. The CSD system is one of three Fe-S cluster biogenesis systems in E. coli and is comprised of the cysteine desulfurase CsdA, the sulfur acceptor protein CsdE, and the E1-like protein CsdL. The biological role, biochemical mechanism, and protein targets of the system remain uncharacterized. Here we present that the active site CsdE C61 has a lowered pK a value of 6.5, which is nearly identical to that of C51 in the homologous SufE protein and which is likely critical for its function. We observed that CsdE forms disulfide bonds with multiple proteins and identified the proteins that copurify with CsdE. The identification of Fe-S proteins and both putative and established Fe-S cluster assembly (ErpA, glutaredoxin-3, glutaredoxin-4) and sulfur trafficking (CsdL, YchN) proteins supports the two-pathway model, in which the CSD system is hypothesized to synthesize both Fe-S clusters and other sulfur-containing cofactors. We suggest that the identified Fe-S cluster assembly proteins may be the scaffold and/or shuttle proteins for the CSD system. By comparison with previous analysis of SufE, we demonstrate that there is some overlap in the CsdE and SufE interactomes. Keywordsiron-sulfur (Fe-S) cluster biogenesis; iron-sulfur cluster assembly; iron-sulfur cluster biosynthesis; cysteine sulfinate desulfinase (CSD); pK a ; CsdA; CsdE; CsdL; SufE Sulfur trafficking pathways are highly conserved, complex protein machineries that are responsible for extracting sulfur from cysteine and synthesizing the sulfur-containing cofactors and thionucleosides, including thiamin, biotin, the molybdenum cofactor, lipoic acid, S-adenosylmethionine, and iron-sulfur (Fe-S) clusters (reviewed in Mueller 2006) 1 . The first step of sulfur trafficking is removal of inorganic sulfur from free cysteine by a cysteine desulfurase, followed by the transient and covalent binding of the sulfur atom to a reactive cysteine residue sulfhydryl within the desulfurase active site, thereby forming a persulfide (R-S-SH) intermediate. Due to the toxicity of free sulfide, the persulfide intermediate is the universal sulfur donor in these pathways. The sulfur atom is then transferred between nucleophilic cysteine residue sulfhydryls on various protein carriers, * Author to whom correspondence should be addressed: Department of Environmental Toxicology University of California, Davis One Shields Ave 4138 Meyer Hall Davis, CA 95616 Phone: 530-754-2271 Fax: 530-752-3394. SUPPORTING INFORMATION AVAILABLE Table S1 (Fig. 3C), Table S2 (Fig. 4A), and Table S3 (Fig. 4A) contain proteomic parameters for the proteins identified. Table S1 ( Fig. 3C) contains accession numbers, molecular weight, protein score, number nonredundant peptides, protein score confidence interval, and sequence coverage. Table S2 (Fig. 4A) groups the proteins by function, presents protein molecular weight, and identifies the proteins containing Fe-S clusters. Table S3 (Fig. 4A) contains acc...

Structural Changes during Cysteine Desulfurase CsdA and Sulfur Acceptor CsdE Interactions Provide Insight into the trans-Persulfuration

“…Thus, the structural analysis of CsdA-CsdE further supports the notion that CsdA/CsdE and SufS/SufE work similarly and that the proteins may have been derived from a common ancestor. Thus, CsdE and SufE likely share some of the protein interactomes as demonstrated by others (36).…”

Global gene expression in Escherichia coli, isolated from the diseased ocular surface of the human eye with a potential to form biofilm

“…Apart from this, upregulation of several genes encoding for toxin production and secretions ( sat, yjg K, chp S, chp B and ygj N) was observed in ocular E. coli biofilm cells which is an important factor that governs the virulence in pathogenic bacterial cells. Virulence of ocular E. coli may also be related to up regulation of Lipid A moiety of LPS, a potent stimulator of the immune system and a trigger of intense inflammation in the host cells [61]. Many genes encoding for proteins that influence antigen presentation ( wzz B and waa L), extracellular matrix remodeling ( yii X), transport of aminoacids and other metabolites ( cbr B, cbr C, his I and mgl B) are also up regulated in ocular E. coli in biofilm stage and may have a role to play in virulence.…”

“…Many genes encoding for proteins that influence antigen presentation ( wzz B and waa L), extracellular matrix remodeling ( yii X), transport of aminoacids and other metabolites ( cbr B, cbr C, his I and mgl B) are also up regulated in ocular E. coli in biofilm stage and may have a role to play in virulence. Costa et al [61] have recently suggested that in E. coli, during biofilm formation on abiotic surfaces, cells are exposed to several DNA damaging agents against which the cells need to be protected. In the present study gene ira D, uvr D and rec C involved in DNA damage repair are up regulated and could be acting to ensure survival of cells faced with oxidative damage.…”

The Escherichia coli BolA Protein IbaG Forms a Histidine-Ligated [2Fe-2S]-Bridged Complex with Grx4

“…52 Along these lines, E. coli Grx4 has been shown to form a disulfide with the sulfur trafficking proteins CsdE and SufE in vivo , linking Grx4 function with regulation of sulfur trafficking. 59, 60 Thus, IbaG/BolA may potentially regulate the activity of Grx4 via binding and/or blocking of the redox-active cysteine.…”