BIOL368/F14:Nicole Anguiano Week 7

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Contents

Preparation for Week 8 Journal Club

The article being used is: Huang, C. C., Tang, M., Zhang, M. Y., Majeed, S., Montabana, E., Stanfield, R. L., Dimitrov, D. S., Korber, B., Sodroski, J., Wilson, I. A., Wyatt, R., & Kwong, P. D. (2005). Structure of a V3-containing HIV-1 gp120 core. Science, 310(5750), 1025-1028. DOI: 10.1126/science.1118398

Vocabulary

  1. Mutagenesis: "Act of mutating by deleting or changing the nucleotide sequence" (Biology Online).
  2. Antigenic: "Having the properties of an antigen (allergen)" (Biology Online).
  3. Angstrom: "A unit of length equal to 0.1 nanometer or 1×10−10 meters, mainly used for expressing sizes of atoms, lengths of chemical bonds, and wavelengths of electromagnetic radiation" (Biology Online).
  4. Glycosylation: "The process of adding sugar units such as in the addition of glycan chains to proteins. An occurrence where a carbohydrate is added to a protein molecule, which can occur in the golgi apparatus" (Biology Online).
  5. Motif: "The smallest group of atoms in a polymer that, when under the influence of a rotation-translation operator, will assemble the rest of the atoms in the chain" (Biology Online).
  6. Monoclonal: " Used of a cell line whether within the body or in culture to indicate that it has a single clonal origin. Monoclonal antibodies are produced by a single clone of hybridoma cells and are therefore a single species of antibody molecule" (Biology Online).
  7. Glycan: "A polysaccharide or oligosaccharide, especially one that is attached to a glucoconjugate, as glycoprotein, glycolipid, and proteoglycan" (Biology Online).
  8. Oligomer: "A molecule made up of a relatively few monomeric subunits" (Dictionary of Genetics Oxford Reference eBook).
  9. Clade: "A monophyletic group; a group of organisms (usually species) that are more closely related to each other than any other group, implying a shared most recent common ancestor" (Biology Online).
  10. Neutralization: "The process of neutralizing a pathogen by antibody acting on the receptors or specific antigen" (Biology Online).

Outline

Introduction

  • The HIV envelope protein is composed of three gp120 proteins and three gp41 proteins
    • It binds to CD4 (via gp120) and then undergoes a conformational change and binds to a coreceptor (CCR5 or CXCR4) in order to enter the host cell.
  • Previous attempts to show the crystal structure of the gp120 protein's viral entry mechanism left out the third variable region (V3).
  • V3 is composed of 31 to 39 amino acids (average being 35), and is extremely important in coreceptor binding. It also determines which coreceptor the envelope protein will bind to.
  • Immune responses against HIV are primarily targeted at V3.

Methods and Results

  • gp120 was difficult to crystalize due to glycosylation and flexibility.
  • Three gp120 proteins with V3 were expressed in Drosophila S2 cells, then complexed with CD4 and the Fab (antigen binding) fragment of X5, a CD4 antibody.
    • The crystallization produced hexagonal crystals, and the structure was ultimately solved using molecular replacement.
  • Figure 1 shows the protein structure of a gp120 protein with V3, bound to CD4 and X5.
    • The structures shown match previously determined protein structures of gp120 (without V3), CD4, and X5.
    • V3 was new, and its structure is clearly displayed.
    • Some differences were observed in gp120 where variations are typically found (N-terminus and variable loops). The regions of gp120 near where V3 had been removed in previous studies remained the same.
    • The heavy chain of the X5 antibody had a movement of up to 17 Å in the third complementarity-determining loop.
  • gp120 is made up of inner and outer domains.
    • V3 comes from the outer domain and is 50 Å long, 15 Å wide, and 5 Å deep.
  • V3 has three structural regions:
    • A conserved base
    • A flexible stem
    • A β-hairpin tip
      • The flexibility and position of the tip varies.
  • Figure 2 gives greater information about V3 split into five parts A-E.
    • A shows the sequences of V3 from two of the three isolates used, JR-FL and HXBc2. The consensus sequences are also shown, with conserved residues in capital letters.
    • B gives the electron density and B values of V3. The image is colored using the B value from blue to red.
    • C shows the entire structure of V3 with the three structural regions labeled (base, stem, tip).
    • D specifically shows the V3 base region.
      • The base is composed of two anti-parallel sheets, one that came from the gp120 core and one that returned to it.
      • The initial three residues are connected via hydrogen bonds, and the side chain of Arg298 hydrogen bonds to three carbonyl oxygens. Pro299</sup> is the first to separate the outgoing and returning strands.
    • E specifically shows the V3 tip.
      • The tip begins as a β-sheet, then has a Gly-Pro-Gly-Arg β-turn. After the turn, the strand is not as well defined.
  • The portions of gp120 important for binding to coreceptors were mapped to two areas
    1. the V3 tip,
    2. the area around the V3 base in gp120, the V3 base itself, and the neighboring residues in that area.
    • The regions were conserved in both sequence and structure after analysis.
  • Areas of the V3 stem important for coreceptor binding are separated by areas with medium to high sequence variation.

Discussion

  • The N-terminus and second extracellular loop of the coreceptor may be separated spacially.
    • It is hypothesized that the N-terminus binds to the gp120 core and V3 base while the V3 tip interacts with the second extracellular loop. Support for this hypothesis comes from two areas.
      1. Biochemical studies ahve shown that the binding of the N-terminus of CCR5 is affected by gp120 alterations to the core and V3 base.
      2. Small-molecule inhibitors of HIV entry that bind to the second extracellular loop do not affect HIV viruses with V3 truncations.
  • Figure 3 shows a schematic of how the gp120 trimer and the V3 may interact with the host cell.
    • A shows how V3 looks in the trimer at the surface of the host cell when gp120 has bound to CD4. The V3 tips are pointed downwards towards the cell, towards the coreceptor.
    • B gives a schematic of coreceptor interaction. The CCR5 coreceptors N-terminus is bound to gp120 and the V3 base, and the V3 tip is interacting with CCR5.
  • There is less tolerance for insertions and deletions in the V3 stem than tolerance for sequence changes.
  • When the gp120 protein is bound to CD4, the V3 tips point downwards towards the cell membrane of the target cell.
  • The determine which coreceptor is bound to, there is what is called the 11/25 rule.
    • The virus will use CCR5 unless the 11th or 25th positions of V3 are positively charged. Then they will use CXCR4.
    • V3 sequences are more conserved in CCR5 viruses.
    • Positions 11 and 25 are in the variable region of V3, and are the same distance from the gp120 core.
      • The are separated by 17 Å, indicating they may bind to different areas of a coreceptor.
  • When gp120 binds to CD4 it undergoes conformational changes that cause V3 to protrude.
    • The conformation of the region that V3 is attached to on gp120 does not change much.
  • Immunization with gp120 or gp120/41 generally causes an immune response directed at V3.
  • The Pro-Gly tip of V3 is often conserved.
  • Figure 4 shows how neutralizing antibodies bind to V3.
    • A shows V3 with its structures as shown after being extracted from peptide-anti-VD neutralizing antibody complexes.
    • B shows the antibody accessibility of V3. V3 can be completely surrounded, meaning it has a high degree of accessibility in order to produce an immune response.
  • Due to HIV having mechanisms that prevent it from being bound to antibodies, the exposure of V3 means it can easily generate an immune response.
  • V3's properties allow it to serve as a general molecular hook.
    • It can hook neighboring protomers, increasing resistance to immune response.
    • When the envelope protein is bound to CD4, the V3 tips protrude downward to the cell membrane, perhaps to help initiate binding to the coreceptor.
    • V3 also determines which coreceptor will be used.

Presentation

Journal Club 2 Presentation

Links

Nicole Anguiano
BIOL 368, Fall 2014

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