Michael R. Pina Week 7

Week 7

Presentation

Powerpoint for week 8

Definitions for article

1. oligomeric - In chemistry, an oligomer consists of less than five monomer units (ολιγος, or oligos, is Greek for "a few"), in contrast to a polymer that, at least in principle, consists of an unlimited number of monomers
   • http://en.wikipedia.org/wiki/Oligomer
2. syncitia - syncytium /syn·cy·ti·um/ (sin-sish´e-um) a multinucleate mass of protoplasm produced by the merging of cells.
   • http://medical-dictionary.thefreedictionary.com/Syncitia
3. propensity - an often intense natural inclination or preference
   • http://www.merriam-webster.com/dictionary/propensity
4. glycoslyation - Glycosylation is the enzymatic process that links saccharides to produce glycans, attached to proteins, lipids, or other organic molecules
   • http://en.wikipedia.org/wiki/Glycosylation
5. hybridoma - Hybridoma technology is a technology of forming hybrid cell lines (called hybridomas) by fusing a specific antibody-producing B cell with a myeloma (B cell cancer) cell that is selected for its ability to grow in tissue culture and for an absence of antibody chain synthesis
   • http://en.wikipedia.org/wiki/Hybridoma_technology
6. murine - of or relating to a murid genus (Mus) or its subfamily (Murinae) which includes the common household rats and mice; also : of, relating to, or involving these rodents and especially the house mouse
   • http://www.merriam-webster.com/dictionary/murine
7. Fab - (Science: immunology) fragment of immunoglobulin prepared by papain treatment. Fab fragments (45 kD) consist of one light chain linked through a disulphide bond to a portion of the heavy chain and contain one antigen binding Site. They can be considered as univalent antibodies.
   • http://www.biology-online.org/dictionary/Fab
8. R-values - In crystallography, the R-factor (sometimes called residual factor or reliability factor or the R-value or RWork) is a measure of the agreement between the crystallographic model and the experimental X-ray diffraction data. In other words, it is a measure of how well the refined structure predicts the observed data.
   • http://en.wikipedia.org/wiki/R-factor_(crystallography)
9. centrosymmetric crystal - The term centrosymmetric, as generally used in crystallography, refers to a space group which contains an inversion center as one of its symmetry elements. In such a space group, for every point (x, y, z) in the unit cell there is an indistinguishable point (-x, -y, -z). Crystals with an inversion center cannot display certain properties, such as the piezoelectric effect
   • http://en.wikipedia.org/wiki/Centrosymmetry
10. Ramachandran plot - A Ramachandran plot (also known as a Ramachandran map or a Ramachandran diagram or a [φ,ψ] plot), developed by Gopalasamudram Narayana Ramachandran and Viswanathan Sasisekharan is a way to visualize dihedral angles ψ against φ of amino acid residues in protein structure.[1] It shows the possible conformations of ψ and φ angles for a polypeptide.
    • http://en.wikipedia.org/wiki/Ramachandran_plot
11. canonical - Basic, canonic, canonical: reduced to the simplest and most significant form possible without loss of generality, e.g., "a basic story line"; "a canonical syllable pattern."
    • http://en.wikipedia.org/wiki/Canonical

Article Outline

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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