BC530 Protein Structure II - UW Courses Web BC530wh - 01 - Seven... · PDF...

Click here to load reader

  • date post

    02-Sep-2019
  • Category

    Documents

  • view

    2
  • download

    0

Embed Size (px)

Transcript of BC530 Protein Structure II - UW Courses Web BC530wh - 01 - Seven... · PDF...

  • 1

    BC530

    Protein Structure II “Seven Levels – part II”

    Fall Quarter 2011

    Wim G. J. Hol

    www.bmsc.washington.edu/WimHol

    PROTEIN STRUCTURE HIERARCHY

    THE SEVEN LEVELS

    7

    6

    5

    4

    3

    2

    1

    0

    MULTI-MACROMOLECULAR ASSEMBLIES

    MULTI-SUBUNIT COMPLEXES

    MULTI-DOMAIN PROTEINS

    DOMAINS

    MOTIFS

    BASIC FOLDS

    CHAINS

    BUILDING BLOCKS

    7

    6

    5

    4

    3

    2

    1

    0

  • 2

    1. Morphological function The cell needs fibers, rings, cages

    2. Cooperative function Binding ligands in one subunit can affect conformation of other subunits in the complex – “allosteric proteins”

    3. Stability against denaturation (???) This is probably a weak argument – small proteins can be very stable indeed – the authors have missed the point that not stability but functioning-with-flexibility is the key property of a protein in living organisms

    4. Reduction of surface area The authors make an interesting case that smaller proteins have a larger surface and hence bind more water molecules per Dalton than multimeric proteins – since a cell contains about 20% protein in volume – proteins cannot all be small since then there would not be enough space left for water!

    Goodsell and Olson give in their review the following reasons for building large protein assemblies:

    D.S Goodsell and A J Olson “Functional Symmetry and Protein Function” Annu. Rev. Biophys. Biomol. Struct. 29, 105-153 (2000)

    Proteins

    Level 6: “MULTI-SUBUNIT PROTEINS”

  • 3

    6.1 Multimers of identical subunits

    6.1.1 Point group symmetry of which there are three types: - “cyclic” - “dihedral” - "cubic": tetrahedral, octahedral and icosahedral

    6.1.2 Helical symmetry Defined by a rotation about the helix axis accompanied by a translation along that axis.

    6.1.3 Non-symmetric Sometimes the deviations from ideal symmetry are small and the term "pseudosymmetry" is used.

    MULTI-SUBUNIT PROTEINS ("MULTIMERS")

    Cyclic Point groups

    Dihedral Point Group Symmetry

    Cubic Point group Symmetry

    7

    7

    The Three Types of Point Group Symmetry

  • 4

    C2 Dimers - Triosephosphate isomerase

    - Lipoamide dehydrogenase

    - and very many others

    C3 Trimers - Influenza virus haemagglutinin

    C4 Tetramers - Influenza virus neuraminidase

    C5 Pentamers - The B subunits of cholera toxin

    C6 Hexamers - Many, including ATPases

    C7 Heptamers - GroES, a chaperonin

    C11 Eleven-mers - Tryptophan RNA-binding Attenuation Protein

    Multi-subunit proteins with Cyclic Point Group Symmetry

    Be aware that not all Dimers of identical subunits need to have C2 Point group symmetry.

    The two subunits can have different conformations; or are related by a non-twofold operation

    A Dimer with Cyclic C2 Point Group Symmetry

    Schematic

    2 subunits

  • 5

    A Heptamer with Cyclic C7 Point Group Symmetry

    GroES The “cap” of a protein folding machine

    Side view Top view

    Shekhar Mande

    7 subunits

    Multi-subunit proteins with Dihedral Point Group Symmetry Definition: One "n-fold axis" with n 2-folds perpendicular to the n-fold.

    All symmetry axes intersect in one point.

    Examples:

    D2 (also called "222")

    Tetramers - Hemoglobin (if  considered identical to ß) - Glyceraldehyde phosphate dehydrogenase - many others

    D3 (also called "32")

    Hexamers - Hemocyanin (arthropods) - Insulin

    D4 ("42")

    Octamers - Hemerythrin

    D6 ("62")

    Dodecamers - Glutamine synthase

    D7 ("72")

    14-mers - GroEL - Proteosome

    D17 34-mers - Disks of Tobacco Mosaic Virus

  • 6

    Schematic GroEL

    A 14-mer with Dihedral D7 (or “72”) Point Group Symmetry

    14 subunits

    TETRAHEDRAL ("T" or "23" symmetry)

    12 subunits - Ferritin

    OCTAHEDRAL ("O" or "432" symmetry)

    24 subunits - Cubic core of the PDC* - Small Heat Shock Protein

    ICOSAHEDRAL ("I" or "532" symmetry)

    60 subunits - Dodecahedral Core of the PDC* - Riboflavin Synthase - Small Spherical Virus Capsids

    Examples:

    Multi-subunit proteins with Cubic Point Group Symmetry

    (* PDC= Pyruvate Dehydrogenase Multi-enzyme Complex)

  • 7

    Tetrahedral

    Octahedral

    Icosahedral

    Concepts in Cubic Point Group Symmetry

    Icosahedron

    Dodecahedron

    Octahedron

    Hexahedron

    Tetrahedron

    The E2 core of the pyruvate dehydrogenase multi-enzyme complex (PDC) 60 subunits - viewed along one of the 30 twofold axes

    Tina Izard

    A 60-mer with Icosahedral (or “I”) Cubic Point Group Symmetry

  • 8

    The E2 core of the pyruvate dehydrogenase multi-enzyme complex (PDC) 60 subunits - viewed along one of the twelve 5-fold axes

    Tina Izard

    A 60-mer with Icosahedral (or “I”) Cubic Point Group Symmetry

    Examples - Actin in e.g. muscle

    Steinmetz, M. O., Stoffler, D. & Hoenger, A. (1997). Actin: from cell biology to atomic detail. J. Struct. Biol. 119, 295-320.

    - Tubulin in microtubules Nogales, E., Wolf, S. G., Khan, I. A., Ludueña, R. F. & Downing, K. H. Structure of tubulin at 6.5 Å and location of the taxol-binding site. Nature 375, 424-426. (1995).

    LEVEL 6.1.2

    Multi-subunit proteins with Helical Symmetry

  • 9

    Globular Actin, or G-Actin, is a four-domain protein of ~ 375 amino acid residues. It binds ATP which it can hydrolyze. It also binds calcium or magnesium.

    Its most important property is to be able to assemble, and disassemble, into fibers, called microfilaments in non-muscle cells.

    (Right-hand figure: The large gold sphere in the center indicates bound ATP: the small sphere the Mg ion)

    Three-dimensional structure of an actin subunit

    Steinmetz et al, J. STRUCTURAL BIOLOGY 119, 295–320 (1997)

    Actin microfilament with helical symmetry

    Steinmetz et al, J. STRUCTURAL BIOLOGY 119, 295–320 (1997)

    Filtered electron microscopy image

    Actin Subunits into e.m. image

    Two intertwined strands

    Two right- handed

    intertwined strands

    With 13 subunits per

    turn, and a pitch of 71.5 nm, or 715 Å.

    715 Å

  • 10

    Notes:

    A. IN MANY CASES ASSEMBLIES OF PROTEINS HAVE ONLY A TEMPORARY EXISTENCE.

    For instance:

    (i) Certain hemoglobins only form multimers when oxygenated and are monomers when deoxygenated.

    (ii) In signal transduction, DNA transcription, cell cycle regulation and many other key processes the assembly and disassembly of multi-protein complexes is very carefully regulated.

    B. THE COMPOSITION OF MULTIPROTEIN COMPLEXES CAN BE REGULATED IN A VARIETY OF WAYS.

    For instance by:

    • phosphorylation & dephosphorylation

    • farnesylation and covalent attachment of other fatty acid containing groups

    • binding of GTP and subsequent slow hydrolysis to GDP

    LEVEL 6.2 Multi-subunit proteins with non-identical subunits

    Examples:

    6.2.1 Different subunits but still forming a symmetric particle

    Heterotetramer 2ß2 (C2) - hemoglobin Heterotetramer 2ß2 (C2) - pyruvate dehydrogenase (E1) of the PDC

    6.2.2 Different subunits with partial symmetry

    AB5 heterohexamer - cholera toxin where the B pentamer has C5 symmetry Heterotrimer (2ß) - Lipoamide dehydrogenase (E3) dimer in complex with

    one E2 binding domain (E2BD) of the PDC

    6.2.3 Different subunits with no symmetry

    Heterodimer (ß) - Ras-like protein (rap)•Ras binding domain Heterotrimer (2ß) - Human Growth Hormone•Receptor Complex Dimer of a 13-mer - Cytochrome c Oxidase 13 different subunits RNA polymerase II - 10 different subunits without any symmetry

    Multi-subunit proteins with non-identical subunits

  • 11

    Hemoglobin: An α2β2-tetramer with C2 Symmetry

    Glu 6 is the point of a key mutation changing HbA into HbS, leading to sickle cell anemia

    B pentamer…

    Titia SixmaView along 5-fold axis

    Cholera Toxin: A Pentamer, plus…

  • 12

    Titia SixmaThe A-subunit

    …a Monomer, make…

    Titia Sixma

    Assembly of the AB5 holotoxin

    A-subunitB-pentamer +

    Cholera Toxin &

    Enterotoxin

    Functions: The B-subunit binds to human cell surface receptors. The A-subunit modifies a key human protein inside the cell.

    Active Site

    One of five Receptor

    Binding Sites

  • 13

    The Pyruvate Dehydrogenase Multi-enzyme Complex (PDC)

    A dynamic multifunctional complex which is differently constructed in different organisms but always contains multiple copies of at least three different types of enzymes:

    E1: the pyruvate dehydrogenase contains the co-factor thiamine diphosphate (TDP) E1's can be 2 homodimers or 2ß2 heterotetramers

    E2: the lipoylacetyl transferase the catalytic domain of E2 forms the high symmetry core of the PDC This can be a 24-mer or a 60-mer; differs per species.

    E3: the lipoamide dehydrogenase contains the cofactor FAD E3's are always a dimer.

    The simplest PDC's have a molecular w