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BioInformatics Lab Tuesday, March 9, 2010 Kristoffer Chin Salomon Garcia Michael Pina

Recurring conformation of the human immunodeficiency virus type 1 gp120 V3 loop. Stanfield RL, Ghiara JB, Ollmann Saphire E, Profy AT, and Wilson IA. Virology (2003) Oct 10; 315(1) 159-73. BioInformatics Lab Tuesday, March 9, 2010 Kristoffer Chin Salomon Garcia Michael Pina. Outline.

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BioInformatics Lab Tuesday, March 9, 2010 Kristoffer Chin Salomon Garcia Michael Pina

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  1. Recurring conformation of the human immunodeficiency virus type 1 gp120 V3 loop.Stanfield RL, Ghiara JB, Ollmann Saphire E, Profy AT, and Wilson IA.Virology (2003) Oct 10; 315(1) 159-73 BioInformatics Lab Tuesday, March 9, 2010 Kristoffer Chin Salomon Garcia Michael Pina

  2. Outline • Introduction • Background on HIV Structure • Background on HIV V3 Region • Materials and Methods • Results and Discussion • References

  3. 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

  4. Glycoproteins binding to CD4 T-cells

  5. V3 Region as part of gp120 • V3 important to viral infectivity • Progresses initial infection to AIDS

  6. Introduction to the Stanfield et al. (2003) Study • 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.

  7. Finding a dominant conformation for V3 • 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

  8. 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

  9. Fab Purification • 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

  10. Fab Crystallization procedure • Were 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

  11. Structure determination • 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

  12. Model building and refinement • 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

  13. Structural Analysis • 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.

  14. 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

  15. Figure 1: comparison of V3 peptide conformations determined by X-Ray crystallography and NMR

  16. Figure 2: stereoview of electron density for the V3 peptide bound to Fab 83.1

  17. 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

  18. Figure 3: the structure of the Fab 83.1-V3 peptide complex

  19. Figure 4: Noncanonical loops from Fab 83.1

  20. 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

  21. Figure 5: stereoview of H3 loop conformations from Fabs 83.1, B02C11, and 26-10

  22. 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

  23. 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

  24. Figure 6: comparison of the V3 peptide conformations

  25. Figure 7: the antibody combining site of Fab 83.1

  26. Figure 8: antigen binding sites of Fabs 83.1, 59.1, 58.2, & 50.1

  27. 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

  28. 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

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