Structure and assembly of P-pili: a protruding hinge region used for assembly of a bacterial adhesion filament.

High-resolution structures of macromolecular complexes offer unparalleled insight into the workings of biological systems and hence the interplay of these systems in health and disease. We have adopted a multifaceted approach to understanding the pathogenically important structure of P-pili, the class I adhesion pili from pyelonephritic Escherichia coli. Our approach combines electron cryomicroscopy, site-directed mutagenesis, homology modeling, and energy calculations, resulting in a high-resolution model of PapA, the major structural element of these pili. Fitting of the modeled PapA subunit into the electron cryomicroscopy data provides a detailed view of these pilins within the supramolecular architecture of the pilus filament. A structural hinge in the N-terminal region of the subunit is located at the site of a newly resolved electron density that protrudes from the P-pilus surface. The structural flexibility provided by this hinge is necessary for assembly of P-pili, illustrating one solution to construction of large macromolecular complexes from small repeating units. These data support our hypothesis that domain-swapped pilin subunits transit the outer cell membrane vertically and rotate about the hinge for final positioning into the pilus filament. Our data confirm and supply a structural basis for much previous genetic, biochemical, and structural data. This model of the P-pilus filament provides an insight into the mechanism of assembly of a macromolecular complex essential for initiation of kidney infection by these bacteria.

[1]  G. Schoolnik,et al.  Nucleotide sequence of the papA gene encoding the Pap pilus subunit of human uropathogenic Escherichia coli , 1984, Journal of bacteriology.

[2]  S. Normark,et al.  Gene products specifying adhesion of uropathogenic Escherichia coli are minor components of pili. , 1986, Proceedings of the National Academy of Sciences of the United States of America.

[3]  E. Egelman.  A robust algorithm for the reconstruction of helical filaments using single-particle methods. , 2000, Ultramicroscopy.

[4]  Wei Zhang,et al.  Combining X-Ray Crystallography and Electron Microscopy , 2005, Structure.

[5]  R J Read,et al.  Crystallography & NMR system: A new software suite for macromolecular structure determination. , 1998, Acta crystallographica. Section D, Biological crystallography.

[6]  G. Waksman,et al.  Chaperone Priming of Pilus Subunits Facilitates a Topological Transition that Drives Fiber Formation , 2002, Cell.

[7]  W. Fiers,et al.  Pap pili as a vector system for surface exposition of an immunoglobulin G-binding domain of protein A of Staphylococcus aureus in Escherichia coli , 1993, Journal of bacteriology.

[8]  V. Stojanoff,et al.  X-ray structure of the FimC-FimH chaperone-adhesin complex from uropathogenic Escherichia coli. , 1999, Science.

[9]  A Leith,et al.  SPIDER and WEB: processing and visualization of images in 3D electron microscopy and related fields. , 1996, Journal of structural biology.

[10]  David Eisenberg,et al.  Amyloid-like fibrils of ribonuclease A with three-dimensional domain-swapped and native-like structure , 2005, Nature.

[11]  G. Schoolnik,et al.  Synthetic peptides corresponding to protective epitopes of Escherichia coli digalactoside-binding pilin prevent infection in a murine pyelonephritis model. , 1988, Proceedings of the National Academy of Sciences of the United States of America.

[12]  M. Levitt.  A simplified representation of protein conformations for rapid simulation of protein folding. , 1976, Journal of molecular biology.

[13]  F. Jacob-Dubuisson,et al.  Development of pilus organelle subassemblies in vitro depends on chaperone uncapping of a beta zipper. , 1996, Proceedings of the National Academy of Sciences of the United States of America.

[14]  Lee Makowski,et al.  Structural polymorphism of bacterial adhesion pili , 1995, Nature.

[15]  Roland L. Dunbrack,et al.  Backbone-dependent rotamer library for proteins. Application to side-chain prediction. , 1993, Journal of molecular biology.

[16]  G. Waksman,et al.  Structural basis of chaperone function and pilus biogenesis. , 1999, Science.

[17]  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.

[18]  H. Smith,et al.  Observations on the pathogenic properties of the K88, Hly and Ent plasmids of Escherichia coli with particular reference to porcine diarrhoea. , 1971, Journal of medical microbiology.

[19]  M. Karplus,et al.  Evaluation of comparative protein modeling by MODELLER , 1995, Proteins.

[20]  Chris Sander,et al.  The double cubic lattice method: Efficient approaches to numerical integration of surface area and volume and to dot surface contouring of molecular assemblies , 1995, J. Comput. Chem..

[21]  J. Pinkner,et al.  Haemophilus influenzae pili are composite structures assembled via the HifB chaperone. , 1996, Proceedings of the National Academy of Sciences of the United States of America.

[22]  S Falkow,et al.  Genetics of digalactoside-binding adhesin from a uropathogenic Escherichia coli strain , 1983, Infection and immunity.

[23]  S. Hultgren,et al.  Periplasmic chaperone recognition motif of subunits mediates quaternary interactions in the pilus , 1998, The EMBO journal.

[24]  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.

[25]  H. Smith,et al.  The production of oedema disease and diarrhoea in weaned pigs by the oral administration of Escherichia coli: factors that influence the course of the experimental disease. , 1968, Journal of medical microbiology.

[26]  C. Brinton.  The structure, function, synthesis and genetic control of bacterial pili and a molecular model for DNA and RNA transport in gram negative bacteria. , 1965, Transactions of the New York Academy of Sciences.

[27]  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.

[28]  D. Langs,et al.  On 'globbicity' of low-resolution protein structures. , 1999, Acta crystallographica. Section D, Biological crystallography.

[29]  S. Hultgren,et al.  Chaperone-assisted assembly and molecular architecture of adhesive pili. , 1991, Annual review of microbiology.

[30]  S. Normark,et al.  The Gal(alpha 1-4)Gal-specific tip adhesin of Escherichia coli P-fimbriae is needed for pyelonephritis to occur in the normal urinary tract. , 1994, Proceedings of the National Academy of Sciences of the United States of America.

[31]  Localization of a critical interface for helical rod formation of bacterial adhesion P-pili. , 2005, Journal of molecular biology.

[32]  A. Craiu,et al.  DNA sequences of three papA genes from uropathogenic Escherichia coli strains: evidence of structural and serological conservation , 1991, Infection and immunity.