title: Physical and functional interactions of a monothiol glutaredoxin and an iron sulfur cluster carrier protein with the sulfur-donating radical S-adenosyl-L-methionine enzyme MiaB.
abstract: The biosynthesis of iron sulfur (FeS) clusters, their trafficking from initial assembly on scaffold proteins via carrier proteins to final incorporation into FeS apoproteins, is a highly coordinated process enabled by multiprotein systems encoded in iscRSUAhscBAfdx and sufABCDSE operons in Escherichia coli. Although these systems are believed to encode all factors required for initial cluster assembly and transfer to FeS carrier proteins, accessory factors such as monothiol glutaredoxin, GrxD, and the FeS carrier protein NfuA are located outside of these defined systems. These factors have been suggested to function both as shuttle proteins acting to transfer clusters between scaffold and carrier proteins and in the final stages of FeS protein assembly by transferring clusters to client FeS apoproteins. Here we implicate both of these factors in client protein interactions. We demonstrate specific interactions between GrxD, NfuA, and the methylthiolase MiaB, a radical S-adenosyl-L-methionine-dependent enzyme involved in the maturation of a subset of tRNAs. We show that GrxD and NfuA physically interact with MiaB with affinities compatible with an in vivo function. We furthermore demonstrate that NfuA is able to transfer its cluster in vitro to MiaB, whereas GrxD is unable to do so. The relevance of these interactions was demonstrated by linking the activity of MiaB with GrxD and NfuA in vivo. We observe a severe defect in in vivo MiaB activity in cells lacking both GrxD and NfuA, suggesting that these proteins could play complementary roles in maturation and repair of MiaB.
authors.affiliation: Life Sciences Division, Lawrence Berkeley National Laboratory, Berkeley, California 94720.
authors.forename: Sylvain
authors.initials: S
authors.lastname: Boutigny
authors.affiliation: Life Sciences Division, Lawrence Berkeley National Laboratory, Berkeley, California 94720.
authors.forename: Avneesh
authors.initials: A
authors.lastname: Saini
authors.affiliation: Physical Biosciences Division, Lawrence Berkeley National Laboratory, Berkeley, California 94720; Joint BioEnergy Institute, Emeryville, California 94608.
authors.forename: Edward E K
authors.initials: EEK
authors.lastname: Baidoo
authors.affiliation: Life Sciences Division, Lawrence Berkeley National Laboratory, Berkeley, California 94720.
authors.forename: Natasha
authors.initials: N
authors.lastname: Yeung
authors.affiliation: Physical Biosciences Division, Lawrence Berkeley National Laboratory, Berkeley, California 94720; Joint BioEnergy Institute, Emeryville, California 94608; Department of Chemical Engineering, University of California, Berkeley, California 94720; Department of Bioengineering, University of California, Berkeley, California 94720.
authors.forename: Jay D
authors.initials: JD
authors.lastname: Keasling
authors.affiliation: Life Sciences Division, Lawrence Berkeley National Laboratory, Berkeley, California 94720. Electronic address: gpbutland@lbl.gov.
authors.forename: Gareth
authors.initials: G
authors.lastname: Butland
mesh_terms: D002384:Catalysis; D002942:Circular Dichroism; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism*; D029968:Escherichia coli Proteins / Q000235:genetics / Q000378:metabolism*; D005609:Free Radicals; D054477:Glutaredoxins / Q000235:genetics / Q000378:metabolism*; D007506:Iron-Sulfur Proteins / Q000235:genetics / Q000378:metabolism*; D009154:Mutation; D025941:Protein Interaction Mapping; D012343:RNA, Transfer / Q000378:metabolism; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D012436:S-Adenosylmethionine / Q000378:metabolism; D013466:Sulfurtransferases / Q000235:genetics / Q000378:metabolism*; D020349:Surface Plasmon Resonance
chemical_list: D029968:Escherichia coli Proteins; D005609:Free Radicals; D054477:Glutaredoxins; C568707:GrxD protein, E coli; D007506:Iron-Sulfur Proteins; C529800:NfuA protein, E coli; D011994:Recombinant Proteins; D012436:S-Adenosylmethionine; D012343:RNA, Transfer; C499584:MiaB protein, E coli; D013466:Sulfurtransferases
keywords: Carrier Protein; Escherichia coli; Gene Knockout; Iron Sulfur Cluster Biosynthesis; Iron-Sulfur Protein; Monothiol Glutaredoxin; Protein-Protein Interactions; Radical SAM Enzyme; Recombinant Protein Expression; S-Adenosylmethionine (SAM)
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