EMBL-EBI The pili are polar flexible filaments of about 5.4 nm diameter and 2500 nm average length.
Neisseria gonorrhoeae expressing pili and interacting with epithelial cells.
Model for Type IV pilus biogenesis and dynamics of fiber formation and retraction
EMBL-EBI Forest, K.T., Parge, H.E., Tainer, J.A. Nature , Forest, K.T., Dunham, S.A., Koomey, M., Tainer, J.A. Mol Microbiol , SOURCE: Neisseria gonorrhoeae A fibre forming cell adhesion protein responsible for the virulent attachment Pilin is a subunit of the pilus, a polar flexible filament, which consists of a single polypeptide chain arranged in a helical configuration of five subunits per turn. PDB 1ay2, 1dzo, 1hpw, 1kb7, 1nil, 1pan CATH SCOP d , j ay2 1dzo 1hpw 1kb7 1nil 1pan d j PDB 2PIL / 1AY2
Type IV Pilin Structure and Assembly: X-Ray and EM Analyses of Vibrio cholerae Toxin-Coregulated Pilus and Pseudomonas aeruginosa PAK Pilin L. Craig, R.K. Taylor, M.E. Pique, B.D. Adair, A.S. Arvai, M. Singh, S.J. Lloyd, D.S. Shin, E.D. Getzoff, M. Yeager, K.T. Forest & J.A. Tainer Molecular Cell, 11, 1139–1150, 2003
EM Analysis of TCP Reveals a Three-Start Helix with a 45 Pitch C Bundle of negatively stained TCP filaments D computed Fourier transform of a single filament within the bundle as indicated by the box in (C). E Left-handed representation of a three-start helix with each start shown in a different colour.
Pili are thin, protein tubes The pilus has a shaft composed pilin. At the end of the shaft is the adhesive tip structure having a shape corresponding to that of specific glycoprotein or glycolipid receptors on a host cell Because both the bacteria and the host cells have a negative charge, pili may enable the bacteria to bind to host cells without initially having to get close enough to be pushed away by electrostatic repulsion. Once attached to the host cell, the pili can depolymerize and enable adhesions in the bacterial cell wall to make more intimate contact.
EMBL-EBI Type IV pili are not merely passive sticky fibres but dynamic machines that participate in a surprising number of functions including: Bacterial aggregation Adhesion to host cells Twitching motility Pilus retraction DNA transformation In another bacterial species, motility. Phage receptor in V. cholerae.
EMBL-EBI EMBL UniProt PDB Assembly (MSD) Microscopy still not the full story - GENOME
EMBL-EBI Pilus gene organisation Many copies of pilin gene throughout chromosome Two are functional, pilE1 and pilE2 All other copies are silent Antigenic variation occurs due to recombination (within mini-cassettes)
EMBL-EBI Antigenic variation in N. gonorrhoeae A single cell can give rise to daughter cells expressing structurally and antigenically different pili Gonococcus has the genetic capacity to make as many as a million different pilin variants All able to bind to same host tissues and to cause the same disease symptoms
EMBL-EBI One of the body's innate defences is the ability to physically remove bacteria from the body by: constant shedding of surface epithelial cells coughing, sneezing, vomiting, and diarrhoea removal by bodily fluids such as saliva, blood, mucous, and urine. PILI are the pathogen’s answer to Mankind's physical defence systems
pili enable adhere N. gonorrhoeae to receptors on target epithelial cells and thus colonize and resist flushing by the body.
EMBL-EBI REMEMBER – this all achieved by simple non-covalent forces
EMBL-EBI PDB Entries and X-Ray results 1.Crystal Structure 2.Molecular Structure (covalent) 3.Oligomeric Assembly What has all this got to do with MSD?
EMBL-EBI Protein Stickiness What does this mean? What is the evidence?
EMBL-EBI PDB Xray coordinates PDB entry the deposited coordinates usually consist of the contents of the asymmetric unit: The contents of the ASU define a single copy of the macromolecule The contents of the ASU consist of more than one copy of the macromolecule The contents of the ASU require crystallographic symmetry operations to be applied to generate the complete macromolecule(s) A combination of the above, including multiple copies and required symmetry transformations
A crystal is a periodic arrangement of a motif in a lattice. The motif can be a single atom, a small molecule, a protein or any combination thereof. Often the motif, also referred to as to the 'asymmetric unit', is subjected to a number of symmetry operations yielding differently oriented copies.
EMBL-EBI The combination of all available symmetry operations (point groups plus glides and screws) with the Bravais translations leads to exactly 230 combinations, the 230 Space Groups. Space Groups Kathleen Yardley Lonsdale Carried out a profound and systematic study of the theory of space groups, methods for their determination, and the possibilities of molecular symmetry that are involved (1924, 1936).
EMBL-EBI benzene C6H6C6H6 Covalent bonded
EMBL-EBI Benzene crystallised in Space Group P6/m 6-fold rotation axis Mirror plane
EMBL-EBI Benzene P6/m in the PDB ATOM C1 x1 y1 z1 occupancy 0.5 ATOM H1 x2 y2 z2 occupancy 0.5 Entire atomic contents:
EMBL-EBI The stronger of the two is the hydrogen bond. The weaker is the van der Waal's forces. Both interactions depend on the same fundamental cause, the charge on electrons, and how that results in attraction and repulsion at an atomic level. HELD TOGETHER BY WEAK FORCES
EMBL-EBI The equation of state for gases and liquids Nobel Prize 1910 The origin of the London van der Waals force lies in the instantaneous dipole generated by the fluctuation of electron cloud surrounding the nucleus of electrically neutral atoms. Johannes D. van der Waals
EMBL-EBI All intermolecular attractions are known collectively as van der Waals forces. The various different types were first explained by different people at different times. Dispersion forces, for example, were described by London in 1930; dipole-dipole interactions by Keesom in van der Waals forces
EMBL-EBI Pauling in 1935 was the first to explain the mysterious stickiness of water molecules. The basic principle behind hydrogen bonding is that the electron deficient hydrogen atom of one polar molecule is attracted to the electron rich side of another polar molecule. Hydrogen bonds are somewhat stronger than van der Waal's forces, and require two components: a donor group and an acceptor group. Hydrogen Bonds
EMBL-EBI Hydrogen Bonds
EMBL-EBI Quaternary Structure Quaternary Structure is defined as that level of form in which units of tertiary structure aggregate to form homo- or hetero-multimers. Consideration of the presence of a quaternary state is important in the understanding of a protein's biological function.
EMBL-EBI Proteins don’t do this – pack by translationals
EMBL-EBI There are three main types of symmetry: symmetry with respect to a plane (mirrors) symmetry with respect to a line (rotations) symmetry with respect to a point (inversions) Symmetry
EMBL-EBI symmetry with respect to a line (rotations) symmetry with respect to a plane (mirrors) symmetry with respect to a point (inversions) Symmetry
EMBL-EBI 1, 2, 3, 4, 6 -fold rotational symmetry These are the only rotational symmetries that can exist in crystals; all others are disallowed. These five rotational axes are called the five Proper Axes Symmetries showing 5-, 7-, 8-, 9-, 10-, 11-, & 13- fold rotations are known for biological molecules – these are observed in the Asymmetric Unit. Rotational symmetry
1g8h Applying 1 st 3-fold Rotation A A’ Residues of Chain A in interface
A A’ Residues of Chain A’ in interface
Applying 2 nd 3-fold Rotation A A’ A”
Also has a 2-fold rotation
Final Assembly is a Hexamer from 23 symmetry
EMBL-EBI If you add translations to rotation axes, you form what are call screw axes. For an nm screw axis, the rotational component is 360/n degrees, and the translations is m/n of the unit translation along the axis. In Biological Crystallography --> Polymers Helices are improper Screw axes – e.g. DNA Screw Axes
EMBL-EBI YopM is a strongly acidic protein containing 13–20 repeats of a 19-residue leucine-rich-repeated motif (LRR). YopM has a crescent shape, formed from parallel β-sheets,with a loose amino terminus95. Four YopM monomers form a hollow cylinder with an inner diameter of 35 Å. YopM is an important virulence factor in Yersinia infection Screw Axes
EMBL-EBI 1jl5 YOPM
4-fold screw axis
EMBL-EBI Also has a 2-fold rotation – infinite cylinder in crystal
1FOU Connector Protein From Bacteriophage Phi29
EMBL-EBI Molecular assembly acts as a mechanical attachment flange between the head and tail units 1FOU
EMBL-EBI 24 Genes give proteins in the Head+Whiskers/Neck 22 Genes give proteins for the Tail+Base Plate 7 Genes give proteins in the Tail Fibres 1 Gene gives the fin attachment protein e.g. in the Head scaffold there are 576 copies of gp22 ALL HELD TOGETHER BY WEAK FORCES Bacteriophage T4
EMBL-EBI Bacteriophage T4 Important Hinge Proteins
EMBL-EBI Most large proteins are built from assemblies of domains that for the most part consist of regions of nearly rigid motions jointed by flexible regions. The activity of many proteins induces conformational transitions by hinge bending, which involves the movement of relatively rigid parts of a protein about flexible joints The conformational switch from open to closed of the flexible loop-6 of triosephosphate isomerase (TIM) The hinge region on the Fc fragment of human immunoglobulin G Hinge-Bending, Swiveling Motions
EMBL-EBI Hinge mechanism that occurs when there is no continuously maintained interface constraining the motion. Hinge motions usually occur in proteins with two domains with one domain rotating about the hinge as a rigid body. The rotation is caused by a few large torsion angle changes within the hinge region. shear mechanism that occurs when two interfaces slide across each other in order to maintain a well- packed interface. Shear motions are typically small so a large shear motion will be composed of a number of individual shear motions. Hinge-Bending, Swiveling Motions
EMBL-EBI Genes Proteins Structure Function
Some Proteins have No predicted structure or regions predicted not to fold Genes Proteins Structure DYNAMICS Function
EMBL-EBI 1e94 M.Bochtler et al, Nature, 403, 800 (2000) Observed Asymmetric Unit
EMBL-EBI 1e separate molecules (?) 2 dodecamers 1 hexamer
EMBL-EBI 1e94 M.Bochtler et al, Nature, 403, 800 (2000)
EMBL-EBI hexamer Grapple
EMBL-EBI 1e94 M.Bochtler et al, Nature, 403, 800 (2000)
EMBL-EBI Heat shock proteins HslV and HslU that form a new ATP-dependent protease in Escherichia coli - ATP-dependent protease complexes rid cells of misfolded or damaged proteins and control the level of certain regulatory proteins. M. Bochtler, C. Hartmann, H.K.Song, G.P.Bourenkov, H.D.Bartunik, and R.Huber (2000) The structures of HSLU and the ATP-dependent protease HSLU-HSLV. Nature 403, 800 Couvreur B., Wattiez R., Bollen A., Falmagne P., Le ray D., Dujardin J.C. (2002). Eubacterial HslV and HslU subunits homologs in primordial eukaryotes.Mol. Biol. Evol. 19,