Robert W Arnold Week 7

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Electronic Lab Notebook 7

Robert W Arnold

Week 7 Assignment

Journal Club 2 - Stanfield et. al 2003

HIV Structure Project

  • New partners assigned.
  • Isaiah and I will be working together to present the Stanfield 2003 paper in Journal Club next week.
  • Began work for Week 8 Journal Club.

Week 8 Journal Club

Word Definitions

  1. lentivirus - a genus of retroviruses that cause persistent infection that typically results in chronic, progressive, usually fatal disease; it includes the human immunodeficiency viruses
  2. oligomeric - a polymer formed by the combination of relatively few monomers
  3. syncitia - A mass of cytoplasm having many nuclei but no internal cell boundaries
  4. rhinovirus - a genus of viruses of the family Picornaviridae that infect the upper respiratory tract and cause the common cold
  5. chimeric - Composed of parts of different origin
  6. hybridoma - a somatic cell hybrid formed by fusion of normal lymphocytes and tumor cells
  7. monoclonal - Of or relating to a protein from a single clone of cells, all molecules of which are the same
  8. hypervariable region - a chromosomal segment characterized by multiple alleles within a population for a single genetic locus
  9. conformation - chemical structure, also called the chemical conformation, the spatial arrangement of atoms and chemical bonds in a molecule
  10. twinning - in crystallography, crystal twinning refers to intergrown crystal forms that display a twin boundary

Journal Club Outline - Stanfield 2003

Introduction

  • HIV-1 is a virus with spikes made of gp120 and gp41 embedded in its outer layer
    • gp120 and gp41 are envelope proteins, both are encoded by gp160
    • gp41 is responsible for staying attached to membrane, which in turn allows for gp120 to bind to cell receptors like CD4 along with coreceptors such as CCR5 and CXCR4
    • gp120 has 5 variable regions, labeled V1-V5
    • the V3 region of gp120 is especially important for HIV-1:
      1. V3 is a loop region with 40 residues at the base of the loop
      2. V3 is able to vary due to cellular environment
      3. it is able to neutralize antibodies
    • Attempts at a vaccine based on the gp120 V3 specific region have been abandoned because they have only been successful in laboratory isolates and small strains
    • Initial viral isolates use the CCR5 coreceptor to infect and are very hard to neutralize by any antibody, including those that target V3
    • The V3 loop can be blocked by interactions within the gp120 complex
    • As the infection progresses to AIDS, V3 regions in gp120 change
      1. V3 acquires more positively charged residues
      2. becomes easier to neutralize, likely because it is more accessible due to conformational change in gp120
    • gp120 changes its conformational state many times
      1. After gp160 codes for gp120 and gp 41
      2. After coreceptor interaction
    • The only crystal structures for gp120 are gp120 core regions from HXB2 and YU2, with some parts of V3 deleted
    • Stanfield et al are trying to determine the different conformational changes of gp120 by studying V3 conformation states and why it is recognized differently by different antibodies
    • they have discovered that certain antibodies recognize one type of V3 conformation while others recognize another type but that both V3 conformations have a B-strand followed by a double B-turn
    • V3 is presumed to be able to adjust its conformation to different antibody binding sites
    • NMR studies have shown V3 peptides are disordered in water solution
    • peptide cyclization, peptide glycosylation, attaching to bacteriophage viral coats, attaching to carrier proteins are all methods that have been used to stabilize V3 loop peptide conformation
    • Stabilized peptides are much more ordered increasing likelihood of GPGRAF region
    • Antibody 83.1 is a group of 5 antibodies
    • They were taken from mice during HIV-1 studies
    • all were raised against a 40-mer peptide containing the V3 sequence from the MN viral isolate
    • These 5 were chosen of 85,000 due to their neutralizing strengths
    • Some monoclonal antibodies are very specific while others are very broad
    • 83.1 and 58.2 neutralize M-tropic isolates

Results and Discussion

  • R-values were higher than expected even with good electron density maps
  • Twinning tests were negative
  • The R-values being higher show a noncrystallographic translation which would result in reflections
  • Table 1 shows average reflection intensity
  • The loops focus in on the V3 region of gp120 and its conformations
  • All of the CDR (complementarity determining region) loops fall into expected classes with the exception of L1
  • The L1 CDR loop tip bends away from the antigen binding site which is unusual
  • When compared with 83.1 it is seen that it moves away from its corresponding site
  • The loop collides with residues such as Pro^H41 and Gly^H42
  • Table 2 summarizes all of the crystallographic data including wavelength, resolution, number of observations, reflections, and completeness
  • CDR H3 has a bulged base that is atypical of its sequence
  • Fab 83.1 is normally able to form a salt bridge between Asp^H101 with a charged residue of Arg or Lys at H94 allowing side chain Trp^H103 will hydrogen bond to the carbonyl oxygen, kinking the base
  • Fab 26-10 and Fab B02C11 also have kinked H3 bases that are not predicted by their sequences
  • 26-10 has its Asp^H101 is stabilized by a hydrogen bond and B02C11 Asp^H101 makes two hydrogen bonds to Asp^H96 and Ala^H99
  • Table 3 shows the sequences of the anti-V3 Fabs 83.1 and 59.1 with CDR sequences in bold
  • weak electron density is seen at both ends of 10-mer peptides
  • the residues at the N-terminus have extended conformation with a type-1 turn around GPGR and then a type-2 turn around GRA
  • different locations of the carbonyl oxygens in 83.1 and 59.1 peptides change torsion angles but the overall shape is the same
  • the peptide contacts with Fab's heavy and light chains,these interactions include:
    1. 7 hydrogen bonds with no charge-charge interactions
    2. 6 hydrogen bonds to peptide main-chain atoms, one of which goes to the Arg^P315
  • The order of the CDR loops which make the most contact from most to least are:
    1. H3
    2. H1
    3. L1
    4. L2
    5. L3
    6. H3
  • Then light chain residues are next, followed by heavy chain residues
  • the conformations of 3 of the peptides, 83.1, 50.1, and 59.1 are all very similar while the fourth, 58.2 has varying residues at the tip of V3
  • The similarly conformed peptides all bind in different ways and locations when binding with the antibody
  • these peptide conformations are thus believed to be the most common or preferential due to the antibody selection for high neutralizing ability
  • the x-ray crystallography is being used to determine all the different conformations that can be used by V3
  • Table 4 shows hydrogen bonding distances between the Fab and the peptide used

Materials and Methods

  • 83.1 was taken from the mice used in early stages of HIV-1
  • Fab fragments were collected, purified, then concentrated to 15.0 mg/ml for crystallization
  • Then it was mixed with a linear peptide
  • crystals grew as clusters of thin plates
  • data was taken as quick as possible because the crystals suffered radiation induced decay
  • a single crystal was used and the data can be seen in Table 2
  • again, noncrystallographic translation was seen between two Fab molecules
  • Fab 59.1 showed strong rotation function with elbow angles around 135 degrees
  • Fab 58.2 was then used as a model to construct only this time with elbow angles like Fab 59.1
  • The hybrid was then mutated to sequence for 83.1
  • all Fab molecules were numbered, light and heavy connections measured, hydrogen bonds counted, and van der Waals interactions counted

Links

Biol 368 Homepage

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