10 The pili are polar flexible filaments of about 5 The pili are polar flexible filaments of about 5.4 nm diameter and 2500 nm average length.
11 Neisseria gonorrhoeae expressing pili and interacting with epithelial cells.
12 Model for Type IV pilus biogenesis and dynamics of fiber formation and retraction
13 PDB 2PIL / 1AY2Forest, K.T., Parge, H.E., Tainer, J.A. Nature , 32-8.Forest, K.T., Dunham, S.A., Koomey, M., Tainer, J.A. Mol Microbiol ,SOURCE: Neisseria gonorrhoeaeA fibre forming cell adhesion protein responsible for the virulent attachmentPilin 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
14 Type IV Pilin Structure and Assembly: X-Ray and EM Analyses of Vibrio cholerae Toxin-Coregulated Pilus and Pseudomonas aeruginosa PAK PilinL. 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. TainerMolecular Cell, 11, 1139–1150, 2003
15 EM Analysis of TCP Reveals a Three-Start Helix with a 45 Pitch C Bundle of negatively stained TCP filamentsD 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.
17 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 cellBecause 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.
18 Type IV pili are not merely passive sticky fibres but dynamic machines that participate in a surprising number of functions including:Bacterial aggregationAdhesion to host cellsTwitching motilityPilus retractionDNA transformationIn another bacterial species, motility.Phage receptor in V. cholerae.
19 EMBLUniProtPDBAssembly (MSD)Microscopystill not the full story - GENOME
20 Pilus gene organisation Many copies of pilin gene throughout chromosomeTwo are functional, pilE1 and pilE2All other copies are silentAntigenic variation occurs due to recombination (within mini-cassettes)
21 Antigenic variation in N. gonorrhoeae A single cell can give rise to daughter cells expressing structurally and antigenically different piliGonococcus has the genetic capacity to make as many as a million different pilin variantsAll able to bind to same host tissues and to cause the same disease symptoms
22 PILI are the pathogen’s answer to Mankind's physical defence systemsOne of the body's innate defences is the ability to physically remove bacteria from the body by:constant shedding of surface epithelial cellscoughing, sneezing, vomiting, and diarrhoearemoval by bodily fluids such as saliva, blood, mucous, and urine.
23 pili enable adhere N. gonorrhoeae to receptors on target epithelial cells and thus colonize and resist flushing by the body.
24 REMEMBER – this all achieved by simple non-covalent forces
25 What has all this got to do with MSD? PDB Entries and X-Ray resultsCrystal StructureMolecular Structure (covalent)Oligomeric Assembly
29 Protein StickinessWhat does this mean?What is the evidence?
30 PDB Xray coordinatesPDB entry the deposited coordinates usually consist of the contents of the asymmetric unit:The contents of the ASU define a single copy of the macromoleculeThe contents of the ASU consist of more than one copy of the macromoleculeThe 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
31 A crystal is a periodic arrangement of a motif in a lattice 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.
32 Space GroupsThe 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.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).
34 Benzene crystallised in Space Group P6/m 6-fold rotation axisMirror plane
35 Benzene P6/m in the PDB Entire atomic contents: ATOM C1 x1 y1 z1 occupancy 0.5ATOM H1 x2 y2 z2 occupancy 0.5
36 HELD TOGETHER BY WEAK FORCES 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.
37 Johannes D. van der Waals The equation of state for gases and liquidsNobel Prize 1910The 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.
38 van der Waals forcesAll 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 1912.
41 Hydrogen BondsPauling 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 andan acceptor group.
43 Quaternary StructureQuaternary 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.
46 Proteins don’t do this – pack by translationals
47 Symmetry 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)
48 Symmetry symmetry with respect to a line (rotations) symmetry with respect to a plane (mirrors)symmetry with respect to a point (inversions)
49 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 AxesSymmetries showing 5-, 7-, 8-, 9-, 10-, 11-, & 13- fold rotations are known for biological molecules – these are observed in the Asymmetric Unit.
50 1g8h A’ A Applying 1st 3-fold Rotation Residues of Chain A in interface
55 Screw AxesIf 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 --> PolymersHelices are improper Screw axes – e.g. DNA
56 Screw AxesYopM 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
68 Bacteriophage T4 24 Genes give proteins in the Head+Whiskers/Neck 22 Genes give proteins for the Tail+Base Plate7 Genes give proteins in the Tail Fibres1 Gene gives the fin attachment proteine.g. in the Head scaffold there are 576 copies of gp22ALL HELD TOGETHER BY WEAK FORCES
69 Bacteriophage T4 Identify genes Identify structures Identify location Becomes Mechanics – just balls and springs
71 Hinge-Bending, Swiveling Motions 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 jointsThe 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
72 Hinge-Bending, Swiveling Motions 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.
90 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, 800Couvreur 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,