Structural basis of pilus subunit recognition by the PapD chaperone.

The assembly of different types of virulence-associated surface fibers called pili in Gram-negative bacteria requires periplasmic chaperones. PapD is the prototype member of the periplasmic chaperone family, and the structural basis of its interactions with pilus subunits was investigated. Peptides corresponding to the carboxyl terminus of pilus subunits bound PapD and blocked the ability of PapD to bind to the pilus adhesin PapG in vitro. The crystal structure of PapD complexed to the PapG carboxyl-terminal peptide was determined to 3.0 A resolution. The peptide bound in an extended conformation with its carboxyl terminus anchored in the interdomain cleft of the chaperone via hydrogen bonds to invariant chaperone residues Arg8 and Lys112. Main chain hydrogen bonds and contacts between hydrophobic residues in the peptide and the chaperone stabilized the complex and may play a role in determining binding specificity. Site-directed mutations in Arg8 and Lys112 abolished the ability of PapD to bind pilus subunits and mediate pilus assembly in vivo, an indication that the PapD-peptide crystal structure is a reflection of at least part of the PapD-subunit interaction.

[1]  R. Houghten,et al.  Generation and use of synthetic peptide combinatorial libraries for basic research and drug discovery , 1991, Nature.

[2]  W. Gaastra,et al.  The nucleotide sequence of the gene encoding the K99 subunit of enterotoxigenic Escherichia coli , 1984 .

[3]  L. Gierasch,et al.  The chaperonin GroEL binds a polypeptide in an alpha-helical conformation. , 1991, Biochemistry.

[4]  M. Ultsch,et al.  Human growth hormone and extracellular domain of its receptor: crystal structure of the complex. , 1992, Science.

[5]  L. Makowski,et al.  Helical structure of P pili from Escherichia coli. Evidence from X-ray fiber diffraction and scanning transmission electron microscopy. , 1992, Journal of molecular biology.

[6]  C. J. Duggleby,et al.  Cloning and nucleotide sequence analysis of the serotype 2 fimbrial subunit gene of Bordetella pertussis , 1987, Molecular microbiology.

[7]  S. Hultgren,et al.  Immunoglobulin-like PapD chaperone caps and uncaps interactive surfaces of nascently translocated pilus subunits. , 1991, Proceedings of the National Academy of Sciences of the United States of America.

[8]  C. Brändén,et al.  Conserved immunoglobulin‐like features in a family of periplasmic pilus chaperones in bacteria. , 1992, The EMBO journal.

[9]  S. Hultgren,et al.  P pili in uropathogenic E. coli are composite fibres with distinct fibrillar adhesive tips , 1992, Nature.

[10]  C. Brändén,et al.  Interactive surface in the PapD chaperone cleft is conserved in pilus chaperone superfamily and essential in subunit recognition and assembly. , 1992, The EMBO journal.

[11]  S. Normark,et al.  Biogenesis of E. coli Pap pili: PapH, a minor pilin subunit involved in cell anchoring and length modulation , 1987, Cell.

[12]  J. Pinkner,et al.  FimC is a periplasmic PapD-like chaperone that directs assembly of type 1 pili in bacteria. , 1993, Proceedings of the National Academy of Sciences of the United States of America.

[13]  Per Falk,et al.  Pilus and nonpilus bacterial adhesins: Assembly and function in cell recognition , 1993, Cell.

[14]  A. F. Williams,et al.  The immunoglobulin superfamily--domains for cell surface recognition. , 1988, Annual review of immunology.

[15]  M. Van Montagu,et al.  Isolation and nucleotide sequence of the F17-A gene encoding the structural protein of the F17 fimbriae in bovine enterotoxigenic Escherichia coli , 1988, Infection and immunity.

[16]  D. Wigley,et al.  Crystal structure of a streptococcal protein G domain bound to an Fab fragment , 1992, Nature.

[17]  S. Clegg,et al.  Identification and characterization of the genes encoding the type 3 and type 1 fimbrial adhesins of Klebsiella pneumoniae , 1989, Journal of bacteriology.

[18]  W. Goebel,et al.  Nucleotide sequence of the sfaA gene coding for the S‐fimbrial protein subunit of Escherichia coli , 1987 .

[19]  L. Forney,et al.  Cloning, expression, and sequence analysis of the Haemophilus influenzae type b strain M43p+ pilin gene , 1990, Infection and immunity.

[20]  E.E. Galyov,et al.  Expression of the envelope antigen F1 of Yersinia pestis is mediated by the product of caf1M gene having homology with the chaperone protein PapD of Escherichia coli , 1991, FEBS letters.

[21]  L. Gierasch,et al.  Different conformations for the same polypeptide bound to chaperones DnaK and GroEL , 1992, Nature.

[22]  Axel T. Brunger,et al.  Extension of molecular replacement: a new search strategy based on Patterson correlation refinement , 1990 .

[23]  J. Sambrook,et al.  Protein folding in the cell , 1992, Nature.

[24]  A. Holmgren,et al.  Crystal structure of chaperone protein PapD reveals an immunoglobulin fold , 1989, Nature.

[25]  J. Hacker,et al.  Analysis of genes coding for the sialic acid‐binding adhesin and two other minor fimbrial subunits of the S‐fimbrial adhesin determinant of Escherichia coli , 1989, Molecular microbiology.

[26]  F. Mooi,et al.  The nucleotide sequence of the gene encoding the K88ab protein subunit of procine enterotoxigenic Escherichia coli , 1981 .

[27]  P. Manning,et al.  Genes for biosynthesis and assembly of CS3 pili of CFA/II enterotoxigenic Escherichia coli: novel regulation of pilus production by bypassing an amber codon , 1989, Molecular microbiology.

[28]  K. Lam,et al.  A new type of synthetic peptide library for identifying ligand-binding activity , 1992, Nature.

[29]  S. Hultgren,et al.  PapD, a periplasmic transport protein in P-pilus biogenesis , 1989, Journal of bacteriology.

[30]  S. Clegg,et al.  Nucleotide sequence and functions of mrk determinants necessary for expression of type 3 fimbriae in Klebsiella pneumoniae , 1991, Journal of bacteriology.

[31]  S. Hultgren,et al.  The PapG adhesin of uropathogenic Escherichia coli contains separate regions for receptor binding and for the incorporation into the pilus. , 1989, Proceedings of the National Academy of Sciences of the United States of America.

[32]  B. Oudega,et al.  The penultimate tyrosine residue of the K99 fibrillar subunit is essential for stability of the protein and its interaction with the periplasmic carrier protein. , 1990, FEMS microbiology letters.

[33]  Kayl,et al.  Characterization of three fimbrial genes, sefABC, of Salmonella enteritidis , 1993, Journal of bacteriology.

[34]  J. Rothman,et al.  Peptide-binding specificity of the molecular chaperone BiP , 1991, Nature.

[35]  S. Falkow,et al.  Nucleotide sequence of pilA, the gene encoding the structural component of type 1 pili in Escherichia coli , 1985, Journal of bacteriology.

[36]  L. Randall,et al.  Peptide binding by chaperone SecB: implications for recognition of nonnative structure. , 1992, Science.

[37]  J. Pflugrath,et al.  Crystal orientation and X-ray pattern prediction routines for area-detector diffractometer systems in macromolecular crystallography , 1987 .

[38]  F. Jacob-Dubuisson,et al.  Outer-membrane PapC molecular usher discriminately recognizes periplasmic chaperone-pilus subunit complexes. , 1993, Proceedings of the National Academy of Sciences of the United States of America.

[39]  F. Jacob-Dubuisson,et al.  Initiation of assembly and association of the structural elements of a bacterial pilus depend on two specialized tip proteins. , 1993, The EMBO journal.

[40]  N. Xuong,et al.  Strategy for data collection from protein crystals using a multiwire counter area detector diffractometer , 1985 .

[41]  F. Lindberg,et al.  Horizontal gene transfer of the Escherichia coli pap and prs pili operons as a mechanism for the development of tissue‐specific adhesive properties , 1992, Molecular microbiology.