title: Influence of the conserved disulphide bond, exposed to the putative binding pocket, on the structure and function of the immunoglobulin-like molecular chaperone Caf1M of Yersinia pestis.
abstract: The Yersinia pestis protein Caf1M is a typical representative of a subfamily of periplasmic molecular chaperones with characteristic structural and functional features, one of which is the location of two conserved cysteine residues close to the putative binding pocket. We show that these residues form a disulphide bond, the reduction and alkylation of which significantly increases the dissociation constant of the Caf1M-Caf1 (where Caf 1 is a polypeptide subunit of the capsule) complex [from a Kd of (4.77+/-0.50)x10(-9) M for the intact protein to one of (3.68+/-0.68)x10(-8) M for the modified protein]. The importance of the disulphide bond for the formation of functional Caf1M in vivo was demonstrated using an Escherichia coli dsbA mutant carrying the Y. pestis f1 operon. In accordance with the CD and fluorescence measurements, the disulphide bond is not important for maintenance of the overall structure of the Caf1M molecule, but would appear to affect the fine structural properties of the subunit binding site. A three-dimensional model of the Caf1M-Caf1 complex was designed based on the published crystal structure of PapD (a chaperone required for Pap pili assembly) complexed with a peptide corresponding to the C-terminus of the papG subunit. In the model the disulphide bond is in close proximity to the invariant Caf1M Arg-23 and Lys-142 residues that are assumed to anchor the C-terminal group of the subunit. The importance of this characteristic disulphide bond for the orchestration of the binding site and subunit binding, as well as for the folding of the protein in vivo, is likely to be a common feature of this subfamily of Caf1M-like chaperones. A possible model for the role of the disulphide bond in Caf1 assembly is discussed.
authors.affiliation: Institute of Immunological Engineering, 142380 Lyubuchany, Moscow Region, Russia.
authors.forename: V P
authors.initials: VP
authors.lastname: Zav'yalov
authors.forename: T V
authors.initials: TV
authors.lastname: Chernovskaya
authors.forename: D A
authors.initials: DA
authors.lastname: Chapman
authors.forename: A V
authors.initials: AV
authors.lastname: Karlyshev
authors.forename: S
authors.initials: S
authors.lastname: MacIntyre
authors.forename: A V
authors.initials: AV
authors.lastname: Zavialov
authors.forename: A M
authors.initials: AM
authors.lastname: Vasiliev
authors.forename: A I
authors.initials: AI
authors.lastname: Denesyuk
authors.forename: G A
authors.initials: GA
authors.lastname: Zav'yalova
authors.forename: I V
authors.initials: IV
authors.lastname: Dudich
authors.forename: T
authors.initials: T
authors.lastname: Korpela
authors.forename: V M
authors.initials: VM
authors.lastname: Abramov
mesh_terms: D000478:Alkylation; D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000502:physiology; D001665:Binding Sites; D002942:Circular Dichroism; D003553:Cystine / Q000737:chemistry / Q000502:physiology*; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D007535:Isomerases / Q000502:physiology; D007700:Kinetics; D008565:Membrane Proteins / Q000502:physiology; D008958:Models, Molecular; D018832:Molecular Chaperones / Q000737:chemistry* / Q000378:metabolism; D008969:Molecular Sequence Data; D010084:Oxidation-Reduction; D011485:Protein Binding; D011487:Protein Conformation; D019704:Protein Disulfide-Isomerases; D011499:Protein Processing, Post-Translational; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000378:metabolism; D013329:Structure-Activity Relationship; D014157:Transcription Factors / Q000378:metabolism; D015010:Yersinia pestis / Q000737:chemistry*
chemical_list: D001426:Bacterial Proteins; C079503:DsbB protein, Bacteria; D008565:Membrane Proteins; D018832:Molecular Chaperones; D011993:Recombinant Fusion Proteins; D014157:Transcription Factors; C070195:CAf1M protein, Yersinia pestis; D003553:Cystine; D007535:Isomerases; D019704:Protein Disulfide-Isomerases