Michael R. Pina Week 7
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Definitions for article
- 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
- syncitia - syncytium /syn·cy·ti·um/ (sin-sish´e-um) a multinucleate mass of protoplasm produced by the merging of cells.
- propensity - an often intense natural inclination or preference
- glycoslyation - Glycosylation is the enzymatic process that links saccharides to produce glycans, attached to proteins, lipids, or other organic molecules
- 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
- 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
- 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.
- 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.
- 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
- 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. It shows the possible conformations of ψ and φ angles for a polypeptide.
- 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."
- 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.