Michael R. Pina Week 7

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Powerpoint for week 8

Introduction

  • HIV has an outer surface of Glycoproteins
    • gp120 responsible for binding to CD4 receptors and coreceptors, CCR5 or CXCR4
    • gp41 anchors gp120 to the viral membrane providing noncovalent association
  • V3 regions have high propensity to elicit neutralizing antibodies
  • V3 region inaccessible due to carbohydrates masking or tertiary or quaternary interactions with gp120 complex
  • Is there a limited range of conformational states that gp120 can adopt?
  • How is V3 loop recognized by antibodies and how an alteration of sequence, conformation, or exposure can affect it.
  • Fabs are antibodies that bind to V3 conformation
  • Fab 83.1, 50.1, and 59.1 bind to a similar conformation of V3 region
  • NMR studies have shown V3 to form similar hairpin loops
  • Stabilization of V3 loops to prevent change of conformation through turns
  • 5 antibodies used for neutralizing and stablization

Results and Discussion

  • Rcryst and Rfree values were slightly higher than other structures determined at 2.6Å resolution
  • Electron density maps were good quality
    • However, repeated refinement and manual rebuilding of the structures caused the higher R-values
    • Rcryst 28.8%, Rfree 32.6%
  • An index (0.45*l) close to an integer value is strong, whereas close to one-half integer is weak
  • All CDR loops fall into their expected canonical classes with the exception of L1
  • The L1 CDR loops have a 5 amino acid insertion after residue L27
  • In both Fabs, the tip of this loop bends away from the antigen binding site in an unusual manner
  • Comparison with other L1 loops shows the angle is about 9Å
  • CDR H3 has a “kinked” base
  • This was not predicted from its sequence
    • At least two other Fabs have kinked H3 bases that were not predicted
  • AspH101 normally forms a salt bridge (with Arg or Lys), but in this case it does not which is unexpected
  • The peptide makes contact with both the light and heavy chains from the Fab
  • 110 total contacts for 1 molecule
    • 7 are hydrogen bonds with no charge-charge interactions
    • 6 hydrogen bonds are to peptide main-chain atoms
  • 1 bond to Arg side chain
  • The H3 CDR makes the most contacts
  • The 83.1 peptide structure is the 4th crystal structure determined for a neutralizing antibody V3 peptide complex
  • Analysis of the 4 peptide reveals that 3 are very similar
    • The 4th differs around the V3 region
  • The 4 antibody peptides were generated from related mice
  • The antibodies themselves do not have structural homology
  • The similarity among the conformation of the peptides is not due to the similarity of the Fabs
  • The peptides, although adopting the same shapes, bind in different orientations and locations in the antibody
  • The antibodies were chosen for ability to neutralize (bind to intact viruses)
  • These peptide conformations should reflect “preferred” conformations of the V3 loop
  • The identified V3 structures represent a recurring conformer on the intact virus
  • The X-rays of V3 peptides in complex with antibodies help define the range of V3 conformation
  • Studies suggest that V3 interacts with coreceptors CCR5 and CXCR4 during cell entry
  • This information may be useful in the design of V3 based inhibitors
  • Ultimately, a better understanding of the gp120/gp41 structure (and the V3 region) is vital for understanding how HIV-1 carries out its binding and fusion activities

Materials and Methods

  • Mab 83.1 was made by immunization of an ASW mice with cyclic peptide RP70
  • Antibody was produced in ascites fluid of a mice and purified with an immobilized protein A column
  • Fab was made from immunoglobin by cleavage
  • Fab was concentrated to 15.0 mg/ml for crystallization studies
  • Fab was mixed with 16-mer peptide MP1 in a 6:1 mole ration
  • Crystals were grown using sitting-drop vapor diffusion method with a reservoir solution of 1.6 M Na/K phosphate, 5% isopropanol, pH 6.0.
    • Crystals grow as clusters of thin plates
    • Crystals used for this experiments grew over a 2-week period
  • FabCrystals ere cryocooled to liquid nitrogen temperatures in order to collect the data in a rapid manner.
  • The crystals were protected by putting them in a solution containing the following 25% glycerol, 1.6 M Na/K phosphate, 5% isopropanol, pH 6.0
  • HKL2000 was used to format all of the data that was obtained
  • To determine dtructure, Matthew coefficient was obtained by two Fab molecules
  • Model was constructed from the constant region of Fab 58.2
  • EPMR program was used to position the model in the cell
    • EPMR also used to locate the first Fab molecule in the asymetric unit
  • TOM/FRODO was used to rebuild the mutated hybrid model and to correct the sequence and were subsequently refined with CNS version 1.1
  • Refinement was carried with tight NCS restraints in the beginning and progressively released towards the end of refinement
  • Kabat convention was used to number the molecules
    • Light and heavy chains are labeled using “L” and “H”
    • Peptide labeled “P” and was numbered according to HXB2 isolate sequence
  • HBPLUS was used to evaluate the Hydrogen bonds
  • Contacsym program was used to assign van der waals contacts.
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