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EMBL-EBI MSD database is structured around the fact that Proteins are “sticky”

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Presentation on theme: "EMBL-EBI MSD database is structured around the fact that Proteins are “sticky”"— Presentation transcript:

1 EMBL-EBI MSD database is structured around the fact that Proteins are “sticky”

2 Classes of cell adhesion molecules cadherins Ig-CAMs selectins integrins

3 EMBL-EBI A short biography of 1 protein whose very existence depends on being as sticky as possible

4 EMBL-EBI EMBL K02078 ttaacgcgta aattcaaaaa tctcaaattc cgacccaatc aacacacccg ataccccatg ccaataaaaa agtaacgaaa atcggcacta aaactgacaa ttttcgacac tgccgccccc ctacttccgc aaaccacacc cacctaaaag aaaatacaaa ataaaaacaa ttatatagag ataaacgcat aaaatttcac ctcaaaacat aaaatcggca cgaatcttgc tttataatac gcagttgtcg caacaaaaaa ccgatggtta aatacattgc atgatgccga tggcaagccc tgaggctttc ccctttcaat taggagtaat tttatgaata cccttcaaaa aggctttacc cttatcgagc tgatgattgt gatcgctatc gtcggcattt tggcggcagt cgcccttccc gcctaccaag actacaccgc ccgcgcgcaa gtttccgaag ccatcctttt ggccgaaggt caaaaatcag ccgtcaccga gtattacctg aatcacggca aatggccgga aaacaacact tctgccggcg tggcatcccc cccctccgac atcaaaggca aatatgttaa agaggttgaa gttaaaaacg gcgtcgttac cgccacaatg ctttcaagcg gcgtaaacaa tgaaatcaaa ggcaaaaaac tctccctgtg ggccaggcgt gaaaacggtt cggtaaaatg gttctgcgga cagccggtta cgcgcaccga cgacgacacc gttgccgacg ccaaagacgg caaagaaatc gacaccaagc acctgccgtc aacctgccgc gataaggcat ctgatgccaa atgaggcaaa ttaggcctta aattttaaat aaatcaagcg gtaagtgatt ttccacccgc ccggatcaac ccgggcggct tgtcttttaa gggtttgcaa ggcgggcggg gtcgtccgtt ccggtggaaa taatatatcg at



7 MSD DATABASE pentamer


9 negatively stained TEM images

10 EMBL-EBI 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 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

14 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

15 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.


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 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.

18 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.

19 EMBL-EBI  EMBL  UniProt  PDB  Assembly (MSD)  Microscopy still not the full story - GENOME

20 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)

21 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

22 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

23 pili enable adhere N. gonorrhoeae to receptors on target epithelial cells and thus colonize and resist flushing by the body.

24 EMBL-EBI REMEMBER – this all achieved by simple non-covalent forces

25 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?

26 EMBL-EBI ChainsResiduesAtomsExp. ResultAssembly MSD Relational Database

27 EMBL-EBI KEY to MSD DataBase

28 EMBL-EBI Biological Context PDB  MSD Oxalate oxidase 1FI2 hexameric

29 EMBL-EBI Protein Stickiness What does this mean? What is the evidence?

30 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

31 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 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).

33 EMBL-EBI benzene C6H6C6H6 Covalent bonded

34 EMBL-EBI Benzene crystallised in Space Group P6/m  6-fold rotation axis  Mirror plane

35 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:

36 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

37 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

38 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

39 EMBL-EBI van der Waals

40 EMBL-EBI Linus Pauling ( ) Nobel Prize 1954 Hydrogen Bonding

41 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

42 EMBL-EBI Hydrogen Bonds

43 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.

44 Crystal Structure

45 Oligomeric Assembly

46 EMBL-EBI Proteins don’t do this – pack by translationals

47 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

48 EMBL-EBI  symmetry with respect to a line (rotations)  symmetry with respect to a plane (mirrors)  symmetry with respect to a point (inversions) Symmetry

49 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

50 1g8h Applying 1 st 3-fold Rotation A A’ Residues of Chain A in interface

51 A A’ Residues of Chain A’ in interface

52 Applying 2 nd 3-fold Rotation A A’ A”

53 Also has a 2-fold rotation

54 Final Assembly is a Hexamer from 23 symmetry

55 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

56 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


58 4-fold screw axis

59 EMBL-EBI Also has a 2-fold rotation – infinite cylinder in crystal

60 Screw Axis

61 EMBL-EBI Screw Axes example tubulins

62 EMBL-EBI Bacteriophage T4 Molecular Machine

63 EMBL-EBI Bacteriophage T4 Identified Gene products

64 Bacteriophage T4 1N7Z Gp8 Baseplate Structural Protein 1QEX Gp9 1EL6 Gp11 Baseplate-tail tube complex 1CZD Gp45 Processivity Clamp 1G31 Gp31 Co-Chaperonin 1OCY Receptor-Binding Domain 1RFO Phagehead Fibritin - whisker antigen control 1C1K Gp59 Helicase Assembly

65 1FOU Connector Protein From Bacteriophage Phi29

66 EMBL-EBI Molecular assembly acts as a mechanical attachment flange between the head and tail units 1FOU


68 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

69 EMBL-EBI Bacteriophage T4  Identify genes  Identify structures  Identify location  Becomes Mechanics – just balls and springs

70 EMBL-EBI Bacteriophage T4 Important Hinge Proteins

71 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

72 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

73 EMBL-EBI Genes  Proteins  Structure  Function

74 Some Proteins have No predicted structure or regions predicted not to fold Genes  Proteins  Structure  DYNAMICS  Function

75 EMBL-EBI FoldIndex© tries to answer to the question: Will this protein fold? It's a dynamic and interactive process that estimates the local and general probability for the provided sequence, under specified conditions, to fold.

76 EMBL-EBI Bacteriophage T4

77 EMBL-EBI Bacteriophage T4 baseplate Emd Entry 1048 Kostyuchenko et al., Nat. Struct. Biol., , 688

78 EMBL-EBI of the tube makes up the hypodermic needle at the tip gp5/gp27 hexamer


80 EMBL-EBI 3-fold Rotation PDB: ASU gp5/gp27 hexamer MSD: Assembly

81 EMBL-EBI PDB Example

82 EMBL-EBI 1e94 M.Bochtler et al, Nature, 403, 800 (2000) Observed Asymmetric Unit

83 EMBL-EBI 1e separate molecules (?) 2 dodecamers 1 hexamer

84 EMBL-EBI 1e94 M.Bochtler et al, Nature, 403, 800 (2000)

85 EMBL-EBI hexamer

86 EMBL-EBI hexamer Grapple

87 EMBL-EBI 1e94 M.Bochtler et al, Nature, 403, 800 (2000)

88 EMBL-EBI dodecamer

89 EMBL-EBI dodecamer

90 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,

91 Easter Island Complex with topknot

92 HSLV (P31059) caps HSLU (P32168) P31059 P32168


94 EMBL-EBI Oligomer type Num Homo Hetero Monomer 7490 dimer 9179(8169) 7215(6261) 1958(1908) trimer 1659(1628) 815( 786) 844( 842) tetramer 2963(2930) 1852(1828) 1111(1102) pentamer 178( 178) 91( 91) 87( 87) hexamer 898( 883) 534( 521) 364( 362) heptamer 53( 52) 29( 29) 24( 23) octamer 421( 418) 209( 208) 212( 210) nonamer 62( 62) 6( 6) 56( 56) decamer 72( 72) 43( 43) 29( 29) undecamer 10( 10) 8( 8) 2( 2) dodecamer 230( 230) 96( 96) 134( 134) STATS on PDB Oligomers ~ 24,000 entries

95 EMBL-EBI Windscreen bug splat examples

96 1fmd


98 EMBL-EBI SYMMETRY Rules –BUT What about -

99 What happened to symmetry? 2:1 hetero-complex

100 EMBL-EBI Insulin/insulin receptor complex

101 The Ribosome – the champion Heterocomplex proteins tossed around the RNA

102 protein aggregates complicate the lives of people who study proteins in vitro

103 Protein Aggregation and Amyloid Diseases - Converting the protein from a soluble to a fibrillar structure

104 EMBL-EBI ACKNOWLEDGEMENTS I have taken from the WWW most of the pictures used here The list of sources is available separately.

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