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

• 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