Ryan N. Willhite Week 7

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 Diffraction data were collected at beamline X4A using phosphorimaging plates and Fuji BAS2000 scanner
 * Protein Production, crystallization and data collection
 * 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

[[Media:JOURNALCLUB.ppt|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.


 * Week 7 Assignment Page