title: Exploring structure and interactions of the bacterial adaptor protein YjbH by crosslinking mass spectrometry.
abstract: Adaptor proteins assist proteases in degrading specific proteins under appropriate conditions. The adaptor protein YjbH promotes the degradation of an important global transcriptional regulator Spx, which controls the expression of hundreds of genes and operons in response to thiol-specific oxidative stress in Bacillus subtilis. Under normal growth conditions, the transcription factor is bound to the adaptor protein and therefore degraded by the AAA+ protease ClpXP. If this binding is alleviated during stress, the transcription factor accumulates and turns on genes encoding stress-alleviating proteins. The adaptor protein YjbH is thus a key player involved in these interactions but its structure is unknown. To gain insight into its structure and interactions we have used chemical crosslinking mass spectrometry. Distance constraints obtained from the crosslinked monomer were used to select and validate a structure model of YjbH and then to probe its interactions with other proteins. The core structure of YjbH is reminiscent of DsbA family proteins. One lysine residue in YjbH (K177), located in one of the α-helices outside the thioredoxin fold, crosslinked to both Spx K99 and Spx K117, thereby suggesting one side of the YjbH for the interaction with Spx. Another lysine residue that crosslinked to Spx was YjbH K5, located in the long and presumably very flexible N-terminal arm of YjbH. Our crosslinking data lend support to a model proposed based on site-directed mutagenesis where the YjbH interaction with Spx can stabilize and present the C-terminal region of Spx for protease recognition and proteolysis. Proteins 2016; 84:1234-1245. © 2016 Wiley Periodicals, Inc.
authors.affiliation: Department of Biochemistry and Structural Biology, Lund University, PO Box 124, Lund, SE-221 00, Sweden.|Department of Biology, Lund University, Sölvegatan 35, Lund, SE-223 62, Sweden.
authors.forename: Yusra
authors.initials: Y
authors.lastname: Al-Eryani
authors.affiliation: Department of Biochemistry and Molecular Biology, University of Southern Denmark, Campusvej 55, Odense M, DK-5230, Denmark.
authors.forename: Morten
authors.initials: M
authors.lastname: Ib Rasmussen
authors.affiliation: Department of Biochemistry and Structural Biology, Lund University, PO Box 124, Lund, SE-221 00, Sweden.
authors.forename: Sven
authors.initials: S
authors.lastname: Kjellström
authors.affiliation: Department of Biochemistry and Molecular Biology, University of Southern Denmark, Campusvej 55, Odense M, DK-5230, Denmark.
authors.forename: Peter
authors.initials: P
authors.lastname: Højrup
authors.affiliation: Department of Biochemistry and Structural Biology, Lund University, PO Box 124, Lund, SE-221 00, Sweden.
authors.forename: Cecilia
authors.initials: C
authors.lastname: Emanuelsson
authors.affiliation: Department of Biology, Lund University, Sölvegatan 35, Lund, SE-223 62, Sweden.
authors.forename: Claes
authors.initials: C
authors.lastname: von Wachenfeldt
mesh_terms: D000595:Amino Acid Sequence; D001412:Bacillus subtilis / Q000235:genetics* / Q000378:metabolism; D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D001665:Binding Sites; D003001:Cloning, Molecular; D003432:Cross-Linking Reagents / Q000737:chemistry; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D015964:Gene Expression Regulation, Bacterial; D005977:Glutarates / Q000737:chemistry; D013058:Mass Spectrometry / Q000379:methods; D009876:Operon; D018384:Oxidative Stress; D010447:Peptide Hydrolases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D010957:Plasmids / Q000737:chemistry / Q000378:metabolism; D011485:Protein Binding; D000072756:Protein Conformation, alpha-Helical; D054730:Protein Interaction Domains and Motifs; D059748:Proteolysis; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D013388:Succinimides / Q000737:chemistry; D013879:Thioredoxins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D014157:Transcription Factors / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism
chemical_list: D001426:Bacterial Proteins; D003432:Cross-Linking Reagents; D005977:Glutarates; D011994:Recombinant Proteins; D013388:Succinimides; D014157:Transcription Factors; C427145:bis(sulfosuccinimidyl)glutarate; D013879:Thioredoxins; D010447:Peptide Hydrolases
keywords: Bacillus subtilis; Geobacillus kaustophilus; protein-protein interaction; proteolytic degradation; redox stress; regulatory proteins; stress response; structure prediction