Stanfield et. al (1999) Outline

Introduction

• HIV-1's exterior membrand is embedded with protein copes of gp120 and gp41. Viral cell entry --> gp120 binds to CD4 and another co receptor, gp41 then fusions the viral --> target cell membranes.
• CXCR4 and CCR5 serve as secondary viral receptors to T-tropic and M-tropic cells.
• Disulfide linked loop is the V3 domain of gp120, 40 amino acids have high degree of sequence diversity. V3=major immunogenic site of virus (termed principal neutralizing determinant.)
• V3 loop exposure varies. Increases when CD4-gp120 interaction. More sensitive to neutralize proteases and antibodies. T-tropic V3 seq. --> more basic charge than M-tropic, due to either side of GPGR tip being positivley charged.
• Examined 245 diff. HIV-1 V-3 loops
  • Some positions of aa are highly variable in aa composition.
  • Near tip of loop --> highly conserved
    • Suggests key fn. and structural role in virus infectivity.
• Disease progression = conversion of primary M-tropic to T-tropic strains. Knowing about conformational loops could help explain changes that take place and coreceptor usage when leading to disease progression.
• Antibodies were let against 40a.a V3 peptide RP70; showed that by preventing viral-cell membrane fusion, it neutralized the virus.
  • Previously thought that these antibodies do not interfere with CD4 binding. Using intact viral gp120 particles in recent studies, show that V3 neutralizing antibodies could prevent HIV-1 binding to CD4+ human cells.
• 50.1 & 59.1 antibody Fab fragments in correlation with V3 peptides. 50.1 - highly specific for MN viral strand. 59.1 - neutralize T-tropic strongly and MN weakly.
• 8a.a. residues from 50.1 were ordered in antibody combining site. Residues were extended beta conformation, two lle residues found in hydrophobic pockets, GPG starting to turn.
  • Torsion angle between epsilon region for Gly residue, torsion angle for PG were type II beta turn.
• 59.1 - 10a.a. residues visible. Five had structural counterparts with 50.1 along with similar mainchain torsion angles.
• Around GPGR, type II beta turn had double bend with type I turn for residues RAFY and GRAF. Arg side chain buried deep in negatively charged pocket.
• Single tight turn around GPGR, found now that it is a much broader double turn. --> double bend around GRAFY & around GPGR, had a single tight turn.
• Ala residue - alpha helical torsion angles of -60 & -45. Similar to Aib.
  • Replace Ala for Aib --> constrain the peptide.
• X-ray structure --> Aib had no sig. peptide conformational changes. Aib not needed for crystallization or binding.
• Restrict conformation of V3 peptides - incorporating hydrazone bond using synthetic hydrogen.
• Fab 58.2 - broadly neutralizing & highly potent antibody
  • Neutralizes M-Tropic and T-Tropic viral strains
  • Structure = two cyclic constrained peptides and Aib-containing peptide

Results and discussion

Structure determination and refinement

• Fab 58.2 structures 1 linear & two hydrazone-linked cyclic peptides (respectively 2.0A & 2.8A).
• Isomorphous structures were determined for crystals of the two Fab-cyclic peptide complexes. Due to single a.a. difference in their peptides.
• Fab molecules identified as L (light chain) and H(heavy chain). BH10 isolate sequence --> how peptides are numbered along with Peptide chain identifier.
  • After P316, two a.a. insert interrupts numbering system.
  • Fab 58.2-Aib142 have Fab residues L1-L212, H1-H230 and P313-P325 peptide residues.
  • Fab 58.2 His-loop -- L1-L212, H1-H230 fab residues w/ hydrazone linker
  • Fab 58.2 Ser-loop -- L1-L211, H1-H230 fab residues w/ cyclic peptide, no hydrazone linker.
• Fab-Aib142 peptide complex structure - 89% residues in favored positions (3 in disallowed regions).
  • Disallowed residues -- 2 found in the constant heavy chain in a disordered loop and 1 residue (Ala L51) that is i+2 in distorted type I turn
• 2.8 A resolution determined 2 fab-cyclic peptide structures, only differ at 1 residue in peptide.
  • His loop complex - 81% residues found in favored positions (w/ 3 in disallowed regions)
  • Ser loop complex - 87% residues found in favored positions (w/ 5 in disallowed regions)