The NCBI accession numbers are in parentheses. Abstract Cell surface pili are polymeric protein assemblies that enable bacteria to adhere to surfaces and to specific host tissues. C-terminal sequences preceding the sortase motif of proven and putative cell wall anchors of pili from S. Identification of intermolecular amide bond by mass spectrometry. The FctB protein was overexpressed in Escherichia coli and purified by immobilized metal ion affinity chromatography and gel filtration as described before The proline-rich tail forms a polyproline-II helix that appears to be a common feature of the basal cell wall-anchoring pilins.
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The latter catalyze covalent polymerization of the major pilins by the formation of intermolecular amide bonds between fcttb C terminus of one subunit fcb a lysine residue on the next 245. Structural superpositions of FctB onto the N-terminal domain of Spy and the B domains of the Staphylococcus aureus Cna protein show that both FctB and the Spy N-terminal domain share a common loop not present in the archetypal CnaB domains. Neither the structure of AP2, nor its mode of attachment to the shaft of the pilus, has as yet been determined, however.
The same PPII-like helix is seen for the proline-rich tails of both molecules of the asymmetric unit, differing only in their overall orientation.
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In many instances, pili are crucial for pathogenesis, because they mediate adhesion and enable colonization of a host 1. Although no structure fctbb yet available for Cpa, tctb is known to have a C-terminal domain homologous with the C-terminal domain of Spy 6 and to be attached to the N-terminal domain of the major pilin using the same lysine that is involved in shaft polymerization The currently available sequence and structural data make it increasingly clear that these Gram-positive pili are assembled in a modular fashion, using pilin subunits with Ig-like domains of shared evolutionary origin, arranged like beads on a string.
The surface model right of the pilus was scaffolded on the crystal structure of fcrb major pilin Spy from S. Such motifs are not apparent in minor pilins, however, nor in some major pilins.
Except for FCT-1, these encode three pilin proteins, of which the major pilin usually designated FctA or backbone pilin BP forms the shaft, with the two minor pilins at the tip and the base; Cpa or ancillary pilin 1 AP1 forms the cap and FctB or ancillary pilin 2 AP2 acts as the cell wall anchor of the pilus 19 The crystal structure of FctB was solved by single isomorphous replacement using in-house diffraction data from SeMet-substituted crystals of FctB.
Atomic coordinates and structure factors have been deposited into the Protein Data Bank www. For the GAS pili, we can build a plausible model, in which the polymeric shaft is generated by the action of a pilus-specific sortase that links a lysine residue from the N-terminal domain of one major pilin subunit to the C terminus of the next.
Water molecules are shown as red spheres. This strain is a T9 serotype fctn its fctA sequence; data not shown but is of unknown M serotype.
Unexpected structural homology between the FctB Ig-like domain and the N-terminal domain of the GAS shaft pilin helps explain the use of the same sortase for polymerization of the shaft and its attachment to FctB.
FCTB – Definition by AcronymFinder
The models of Spy and Spy were then docked onto the structure of the backbone pilin Spy PDB code 3B2Mbased on the pilus-like molecular association seen in crystals of Spy 6. Pro-rich stretches are often pivotal in protein-protein interactions 43and PPII helices at the C terminus of cell wall-anchored proteins could play a role in interaction with other proteins such as chaperones or complexes involved in export or assembly.
Our original goal was to determine the structure of Spy, the basal pilin from the M1 strain SF, for which we had mass spectral data from the native pili. The proline-rich tail forms a polyproline-II helix that appears to be a common feature of the basal cell wall-anchoring pilins. A hinge at Leu at the start of the tail enables the rigid proline-rich region to flex like a lever arm relative to the IgG-like domain.
Thus, SpaB anchors the pilus structure to the cell wall. Refinement statistics are in Table 2.
These data indicate that Spy and other AP2 proteins are the basal pilins for GAS pili and should then be tethered to the cell wall by the housekeeping sortase SrtA Athe fold of FctB, drawn as a schematic diagram. FctB and Spy from S. The construct used encompasses the mature protein from the signal peptidase cleavage site through to the LPLAG sortase cleavage motif.
Peptides were identified manually or with a Mascot search engine version 2. The similarity of the motifs displayed by the tip and backbone pilins is consistent with their recognition by the same sortase, whereas the basal pilins share a more generic LP X TG-type motif, consistent with attachment by a housekeeping sortase. Open in a separate window. Fundamental to understanding Gram-positive pilus assembly are the questions of what structural elements in the pilin subunits determine their role in the pilus, where they are incorporated into the overall assembly and by which sortase.
Dashed lines indicate the location of intermolecular isopeptide bonds. In contrast, much less fctbb known so far of the structural determinants that underlie the incorporation of minor pilins at the tip and the base. FctB Asn aligns precisely with Lys of the Spy isopeptide bond and Gln in FctB replaces Glu, which catalyzes the formation of the isopeptide bond in Spy With the knowledge that Lys of Spy was a likely candidate for the essential lysine, we were able to sort through unassigned peptides from the pilus digest and identify a peptide containing fctn Spy Lys and the C-terminal Thr of Spy Fig.