title: A disulfide bridge network within the soluble periplasmic domain determines structure and function of the outer membrane protein RCSF.
abstract: RcsF, a proposed auxiliary regulator of the regulation of capsule synthesis (rcs) phosphorelay system, is a key element for understanding the RcsC-D-A/B signaling cascade, which is responsible for the regulation of more than 100 genes and is involved in cell division, motility, biofilm formation, and virulence. The RcsC-D-A/B system is one of the most complex bacterial signal transduction pathways, consisting of several membrane-bound and soluble proteins. RcsF is a lipoprotein attached to the outer membrane and plays an important role in activating the RcsC-d-A/B pathway. The exact mechanism of activation of the rcs phosphorelay by RcsF, however, remains unknown. We have analyzed the sequence of RcsF and identified three structural elements: 1) an N-terminal membrane-anchored helix (residues 3-13), 2) a loop (residues 14-48), and 3) a C-terminal folded domain (residues 49-134). We have determined the structure of this C-terminal domain and started to investigate its interaction with potential partners. Important features of its structure are two disulfide bridges between Cys-74 and Cys-118 and between Cys-109 and Cys-124. To evaluate the importance of this RcsF disulfide bridge network in vivo, we have examined the ability of the full-length protein and of specific Cys mutants to initiate the rcs signaling cascade. The results indicate that the Cys-74/Cys-118 and the Cys-109/Cys-124 residues correlate pairwise with the activity of RcsF. Interaction studies showed a weak interaction with an RNA hairpin. However, no interaction could be detected with reagents that are believed to activate the rcs phosphorelay, such as lysozyme, glucose, or Zn(2+) ions.
authors.affiliation: Institute of Biophysical Chemistry and Center for Biomolecular Magnetic Resonance, Goethe University, 60438 Frankfurt/Main, Germany.
authors.forename: Vladimir V
authors.initials: VV
authors.lastname: Rogov
authors.forename: Natalia Yu
authors.initials: NY
authors.lastname: Rogova
authors.forename: Frank
authors.initials: F
authors.lastname: Bernhard
authors.forename: Frank
authors.initials: F
authors.lastname: Löhr
authors.forename: Volker
authors.initials: V
authors.lastname: Dötsch
mesh_terms: D001425:Bacterial Outer Membrane Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D018441:Biofilms / Q000254:growth & development; D018360:Crystallography, X-Ray; D004220:Disulfides / Q000737:chemistry* / Q000378:metabolism*; D004926:Escherichia coli / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D029968:Escherichia coli Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D009154:Mutation; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D015398:Signal Transduction / Q000502:physiology; D013329:Structure-Activity Relationship
chemical_list: D001425:Bacterial Outer Membrane Proteins; D004220:Disulfides; D029968:Escherichia coli Proteins; C078611:rcsF protein, E coli