Ryan N. Willhite Week 7

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Outline

I. Introduction

  • Entry of HIV involves a sequential interaction of
    • the envelope glycoprotein (gp120)
    • CD4 glycoprotein
    • chemokine receptor (primary receptor)
  • CD4i (antibodies that block gp120-CD4 complexes to the chemokine receptor)
  • CCR5 and CXCR4 for HIV-1 (secondary receptors)
  • CD4 binding induces conformational changes in the gp120
  • Entry of HIV is mediated by envelope glycoproteins
  • 5 variable regions
  • Variable and non variable regions are glycosylated
  • V3 loop determines specificity
  • Gp41 (transmembrane coat proteins) variants found in all enveloped viruses
  • N-terminal fusion peptides which participate in membrane fusion
  • Enveloped viruses tend to be characteristic in entry
  • Direct membrane penetration (HIV)
  • HIV causes destruction of CD4 T cells which ultimately leads to AIDS.

II. Purpose of this Study

  • Gp120 glycoprotein has important role in
    • receptor binding
    • interactions with neutralizing antibodies
  • Information about the gp120 structure is important for understanding HIV infection
  • Assist in designing therapeutic strategies.
  • Overall purpose is to observe the mechanism of HIV entry and intervene

III. Determining the Structure

  • Devised a crystallization strategy that modified the protein surface
  • Obtained crystals of
    • a ternary complex composed of a truncated form of gp120
    • the N-terminal two domains (DID2) of CD4
    • Fab from the human neutralizing monoclonal antibody 17
  • The ternary structure was solved by
    • combinations of molecular replacement
    • isomorphous replacement
    • density modification techniques

IV. Methods

  • Protein Production, crystallization and data collection

Diffraction data were collected at beamline X4A using phosphorimaging plates and Fuji BAS2000 scanner

  • Structure determination
    • To locate Fab 17b in the ternary complex crystals, rotational searches with 52 different Fab models were made with program MERLOT*
    • Crystals soaked in over 20 different heavy atom compounds
    • Isomorphous replacement phasing*
    • Automatic concatenation of unmodelled density using PRISM
  • Measure deviation

Powerpoint Presentation

Unfamiliar Words:

  1. glycoprotein-are proteins that contain oligosaccharide chains covalently bound to polypeptide side chains.
  2. chemokine-One of a large group of proteins that act as lures and were first found attracting white blood cells.
  3. cavity-laden-
  4. oligomeric-A polymer that consists of two, three, or four monomers.
  5. virion-A complete viral particle, consisting of RNA or DNA surrounded by a protein shell and constituting the infective form of a virus.
  6. fusogenic-cause cell fusion
  7. interfacial cavities
  8. haemagglutinin-involved in influenza. clogs red blood cells together.
  9. steric occlusion- as a General Pro Region Inhibition Mechanism
  • pubs.acs.org/cgi-bin/jtext?bichaw/36/i13/abs/bi962341o
  1. cryoprotectant- protects biological tissue from freezing.
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