Ryan N. Willhite Week 7

Notes on Kwong Paper (RD Outline)

• Entry of HIV involves a sequential interaction of the envelope glycoprotein (gp120) and CD4 glycoprotein and chemokine receptor. (primary receptor)
• There’s a conformational change among binding
• Overall purpose is to observe the mechanism of HIV entry and intervening
• HIV causes destruction of CD4 T cells which ultimately leads to AIDS.
• Entry is mediated by envelope glycoproteins
• 5 variable regions
• Variable and non variable regions are glycosylated
• CD4 binding induces conformational changes in the gp120

1. Glycoprotein

• • Some of which involve the exposure and/or formation of a binding site for specific chemokine receptors
• CCR5 and CXCR4 for HIV-1 (secondary receptors)
• V3 loop determines specificity
• CD4i (antibodies that block gp120-CD4 complexes to the chemokine receptor)
• Gp41 (transmembrane coat proteins) variants found in all enveloped viruses share similarity in sequences
  • Especially N-terminal fusion peptides which participate in membrane fusion
• Enveloped viruses tend to be distinctive in entry
  • Direct membrane penetration (HIV)
  • Endocytosis (influenza)
• Purpose---Because of the important role of the gp120 glycoprotein in receptor binding and interactions with neutralizing antibodies, information about the gp120 structure is important for understanding HIV infection and for the design of therapeutic and prophylactic strategies.

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