Stanfield et. al (1999) Outline

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Contents

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)

Structure descriptions and comparisons

  • Fab 58.2 --> mouse lgG1 k antibody. Elbow Angles -- 174.3, 144.8, and 144.3 in Aib142 His loop and Ser loop complexes.
  • CDR loops -- L1, L2, L3, and H2 --> canonical classes 5,1,1,1. H1 CDR loop -- 1 a.a insert after 35th residue (canonical class 2) but other H1 loops of same length are N10 and AN02.
    • W/ increasing length of H1 loop, increasing flexibility.
  • Linear Aib142 peptide (24 a.a.'s) - Aib replacing AlaP323 . Extended conformation found for RHI residues that connect to type 1 turn around GPGR.
    • Found Vla turn for GrAibF residues. Type 1 turn around RAibFY residues --> resulting in GRAibFy residues a double bend.
      • Arg (P322) - bound in deep pocket w/ negatively charged antibody site --> form interactions (charge-charge) w/ Asp(L94) and Glu(H95). Have similar conformation to bound portions of peptide.
  • Found 3 peptides with identical orientations bind to antibody, Arg(P322) going into deep (-) charged pocket with peptide on flat Fab surface.
  • 6 CDR loops bind to peptide for Fab. Not used is CDR L2 and CDR H1 (minor contacts).
    • CDR loop pattern is typical for antipeptide and antihapten antibodes.
  • Between Arg(P322) and Asp(L94) and Glu(H95), charge-charge interactions were made. Additional interaction btwn Arg(P313) and Asp(L28) in Aib142 peptide.
  • His and Ser loop resolution --> insufficient to determine placement of water molocules. IN refinement of Aib142 complex, 120 water molocultes added & on Fab-peptide interface, 6 were found buried.
  • Structural results coincide with epitope mapping experiments of 58.2 antibody.

Two Different V3 Conformations

  • Bound to Fab 58.2, V3 peptide conformation notable feature --> differs from previously determined structures w/ same peptide in two Fabs.
  • Determined V3 loop structures:
    • Fab 50.1 - peptide epitope CKRIHIGPG
    • Fab 59.1 - peptide epitope HIGPGRAFYT
  • 3 generated antibodies against RP70
    • 5 residues (HIGPG) bound to both 50.1 & 59.1 Fabs - with low rmsds could be superimposed.
    • For type II beta turn around GPGR, both peptides adopt torsion angles.
  • Peptides bound to Fabs 58.1 and 59.1:
    • Contain beta turn along with double bend
      • For central GPGR residues, diff. torsion angles change character of bends and turns.
  • Other studies show in water, V3 loop is disordered but for GPGR residues has tight turns.

Correlation of Structure with Function

  • Observed:
    • In solution, 2 different conformations for tip of the loop
    • While bound to diff. antibodies, found 2 diff. conformations of V3 loop
  • Biological relevance, antibodies bind to gp120 for neutralization activity.
  • Conformations may or may not be related to viral phenotype or just an indications that the V3 loop is capable to adopt different conformations and is flexible.

Biological Implications

  • HIv-1 - virus of membrane envelope, covered with copes of gp120 and gp41 proteins.
  • V3 loop of gp120 involved in MANY different aspects of virus infectivity -- subject of interest.
  • At the tip of gp120 V3 loop, conservation of Gly-Pro-Gly-Arg/Gln, while surrounded with high sequence diversity regions, implicates conservation of structure is correlated with biological fn.
  • Tropism results from change in one or more a.a. in stem of V3 loop.
  • Not seen if it alters loop structure or at top of the loop, effects a switch in conformation in the Beta turn.
  • Crystal determination of Fab fragments --> towards understanding loop conformational flexibility, but answers to other ?'s are unanswered.
    • Unanswered questions --> further study to reveal biological role of V3 loop.

Materials and Methods

  • Chemical synthesis prepared linear Aib142 peptide. N-terminus was acetylated and Acm group protected Cys side-chain.
  • To cleave peptide from resin, mixture of trifluoroacetic acid was used.
  • Purified peptide using Cosmosil C-18 reverse phase column.
  • Using modified a.a's to form hydrazone link, cyclic peptides were synthesized.
  • Using sitting drop, vapor diffusion method (at 22.5 C), all crystallizations were carried out.
  • Fab 58.2 His and Ser loop complexes, crystals obtained w/ solution.

Figures and Tables

  • Figure 1
    • V3 loop peptide sequences shown. Shows replacement of sequences and outcome of the 40 a.a.'s that were put up against the three fabs.
  • Table 1
    • Shows refinement statistics and x-ray diffraction data for Fab 58.2 complexes. Shows different peptides and their data (Aib142, His loop, Ser loop). Shows calculations for R-crystallization.
  • Figure 2
    • Shows Fab 58.2 x-ray structures. Light chain shown in cyan, heavy chain shown in blue. Red shows all atoms of the bound peptide.
  • Figure 3
    • H1 loop comparisons for Fabs 58.2 shown in red, AN02 shown in blue, and N10 shown in yellow.
  • Figure 4
    • 4 different electron density models for V3 peptides that are bound to Fab 58.2.
    • One Fab 58.2-Aib142 complex shown, show type 1 Beta turn.
  • Figure 5
    • Binding pocket of the Fab 58.2 with Aib142 peptide bound to it. (A)Shows antibody combining sites, positive regions in blue and negative regions in red. (B)Combining site of heavy and light chains.
  • Table 2
    • Between bound peptides and Fab 58.2, shows Van der Waals contacts. All contacted complexes first shown and then individual contacts specific to individual complexes are shown.
  • Table 3
    • Shows salt bridge interactions and hydrogen bonds in Fab 58.2 peptide complexes.
  • Figure 6
    • Shows V3 loop conformations. Superimposed the peptides bound to Fabs 50.1,59.1, and 58.2. Shows similar torsion angles for all four peptides for IHI and AF residues.
  • Table 4
    • Displays different Mainchain dihedral angles of the V3 peptides. 50.1, 59.1, and 58.2 peptides shown.

Presentation

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