BIOL368/F14:Isabel Gonzaga Week 7

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Contents

Preparation for Week 8 Journal Club

Source: 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. Coreceptor: (n.) A receptor molecule on the surface of a cell that enhances the activity of another receptor following binding of its extracellular ligand. (e.g. CD4 proteins for T-cell receptors). Source: A Dictionary of Biology, 6ed.
  2. Fab Fragment: (n.) Short for Fragment Antigen-Binding. ~45kDa protein fragment obtained by papain hydrolysis of immuniglobulin molecule. Conists of one intact light chain linked by disulfide bond to N terminus of contiguous heavy chain. each fragment contains one antigen binding site. Source: Oxford Dictionary of Biochemistry and Molecular Biology, 2ed.
  3. Hairpin: (n.) any part of a linear molecular structure, e.g. of a polynucleotide strand or a prostaglandin, in which two adjacent segments of the molecule are folded back one on the other and held in that conformation by secondary molecular forces such as hydrogen bonds or van der Waals interactions. Source: Oxford Dictionary of Biochemistry and Molecular Biology, 2ed.
  4. Heavy Chain: (n.) The heavier of two types of polypeptide chain found in immunoglobulin molecules. Linked to a light chain and an identical heavy chain, often by disulfide bonds. Source: Oxford Dictionary of Biochemistry and Molecular Biology, 2ed.
  5. Immunodominant: (adj.) The attribute of a part of an epitope that contributes a disprpoportionately greta portion of the binding energy. Source: Oxford Dictionary of Biochemistry and Molecular Biology, 2ed.
  6. Modulation:(n.) The adjustment or regulation of the degree or activity or something; control of the activity of a regulatory enzyme by an effector. Source: Oxford Dictionary of Biochemisry and Molecular Biology, 2ed.
  7. Nuclear Magnetic Resonance: (n.) The absorption of electromagnetic radiation (radio waves) by certain atomic nuclei placed in a strong and stable magnetic field. This results in a change of orientation of the nuclei, which respond to the magnetic field like miniature bar magnets. The main application of NMR is in a form of spectroscopy (NMR spectroscopy) used for chemical and biochemical analysis and structure determination. Source: A Dictionary of Biology, 6ed.
  8. Spike: (n.) an inflorescence, such as the catkin of the pussy willow, in which the flowers arise directly from a central axis, the rachis Source: A Dictionary of Genetics, 8ed.
  9. Trimer (n.) a molecular complex having three components or subunits. If these are identical, it is referred to as a homotrimer; if any one is different from any other, it is called a heterotriemer. Source: Oxford Dictionary of Biochemistry and Molecular Biology, 2ed.f
  10. Truncation (n.) Something shorted by cutting, or terminated prematurely. Source: Oxford Dictionary of Biochemistry and Molecular Biology, 2ed.

Article Outline

Introduction

  • HIV envelope spike allows for receptor binding and virus entry
  • Trimeric spike contains 3 gp120 exterior and 3 gp41 transmembrane glycoproteins
  • Virus entry strategy: CD4 binds to the gp120 protein and induces a conformational change, which causes binding to coreceptor (CCR5 or CXCR4) and allows virus to enter
  • Reason for research: Previous work has not included the 3rd Variable Region (V3) in analyzing the structure of the gp120 core in the coreceptor binding site
  • V3 contains ~35 amino acids
    • Critical for coreceptor binding, determines which coreceptor (CCR5 or CXCR4)
    • Interacts to controls overall viral sensitivity to neutralization
    • Immunizing HIV-1 envelope glycoprotein responses directly target V3 region for neutralization
  • Main Article Findings:
    • V3 is a 'molecular hook' that snares coreceptors and modulates subunit associations in spike
    • Findings are compatible with immunodominant response

Methods/Results

  • Variational crystallization and technologies from structural genomics were used to obtain crystals for x-ray structural analysis, as current gp120 structure inhibits crystallization
  • Gp120 core with V3 from three Clade B isolates were made and expressed in Drosophilia S2 cells
  • Proteins were isolated, deglycosylated, purified and complexed iwth CD4 and a CD4-induced antibody
  • 13 different complexes were screened robotically and crystallization was optimized manualy
    • gp120 with V3 from JR-FL formed hexagonal crystals (diffracting 3.5 angstroms with x-rays from Advanced Photon Sourced undulator beam line) when attached to CD4 and antigen-binding fragment of X5 antibody
  • Structure determined using molecular replacement
    • Overall CD4 + X5 + core GDP resembled the individual structures
      • Core gp120 had differences in variable loops, and at N terminus
      • V3 base not affected by previous truncations (original structure in tact)
      • Third complementarity-determining loop in X5 heavy chain (CDR H3) had large structural change; alpha C move up to 17 angstroms (which is one of the largest induced fits observed for an antibody)
Figures and Tables
  • Figure 1:
    • The deduced structure of the HIV-1 gp120 core assembled with V3 (red), CD4 receptor (yellow), and Fab portion of X5 antibody (blue). Image is oriented so viral membrane is at top and the immune cell is towards the bottom.
  • Figure 2:
    • A: sequence comparison between JR-FL and HXBc2 and consensus sequences of clades A, B and C.
    • B: V3 map of electron density and B values. Blue denotes lower atomic mobility and red denotes higher mobility.
    • C: Ball and stick structure of V3 is shown and labeled with base, stem and tip regions.
    • D: Image of V3 base, extending the antiparallel street and interacting on the outer domain of gp120. Patterns of stabilizing Hydrogen bonds are displayed here. Three residues hydrogen bond, then interactions are interrupted by conserved residues, Arg 298 (which hydrogen bonds to three carbonyl oxygens) and Pro 299 (which initiates the separation of strands). Arg and Pro are highlighted in yellow, while the disulfide is highlighted in orange.
    • E: the downward β-conformation of the V3 tip which turns at the Gly-Pro-Gly-Arg turn. After this mark, there is less denisty and greater disorder.
  • Figure 3:
    • A: V3 imaged with trimer at target cell surface (towards bottom of page). The complex structure was superimposed to the structure of four domain CD4, and the trimer model was obtained by analyzing surface parameters.
    • B: CCR5 (green) pictured with N terminus reaching towards the gp120 molecule. V3 (red) has a conserved based that interacts with the N terminus of the coreceptor, while it's legs allow the tip to reach downward towards the second extracelluarl loop in CCR5.
  • Figure 4:
    • A: V3+Core gp120 shown as extracted from peptide-anti-V3 neutralizing antibody complexes after superpositon of the V3 tip
    • B: Core gp120+V3 is shown as ribbons. Fab fragments bind at the coreceptor binding sites on core or V3, surrounding core iwth neutralizing antibodies and showing it's high degree of accessibility and ability to generate immune responses

Discussion

  • Three structural regions of V3
    1. Conserved base
    2. Flexible stem
    3. β-Hairpin tip
      • Flexibilty likely due lattice contact (H bonds between V3 backbone between Isoleucines at amino acid positions 307 and 309)
  • gp120: 2 main features for coreceptor binding (V3 Tip and gp120 core) had conserved sequences and structure
    • Consistent with Stanford et al (2009) - reported recurring conformations for V3 tip in antibody peptide complexes
    • Conserved regions critical for receptor binding are 10-20angstroms apart, and separated by V3 variability portions in stem
  • Suggest that N terminus of the coreceptor will reach to bind to the core and V3 base, and V3 tip of gp120 reaches downward towards the second extracellular coreceptor loop
    • Supported by several resources
      • Cormier et al. (2002) showed that gp120 alterations at the core or V3 base affect CCR5 N-terminal peptide bindings to gp120
      • Lin et al. (2005) showed that mutant viruses with V3 truncations are no longer affected by small molecule HIV inhibitors that bind to the second extracellular loop of the coreceptor
  • Also suggest the V3 stem is less able for mutations via insertions or deletions, compared to other gp120 variable loops
  • Amino Acids at the 11th and 25th positions of V3 determine which coreceptor binds
    • if positive: CXCR4. Otherwise: CCR5 (more conserved)
    • Positions within variable stem; separated by alpha C distance of 17 angstrom
      • Positions 11 and 25 recognize different portions of the coreceptor
  • Liganded (with CD4) vs unliganded structures of gp120 were compared to check for reliability of extended structure that was observed, found that local conformation remained relatively the same
  • Reasons for V3 directed immunodominant response
    • Pro-Gly tip conserved in V3 peptides. Superimposing this with the V3 tip in the core and structure allows for V3 peptide-binding antibodies to completely surround V3
    • Additionally, mechanisms hide the rest of the HIV envelope from antibody binding
  • V3 as a general molecular hook
    • Attributes: high surface area, chemically reactive backbone, conformational flexiblity, extended nature
    • Before binding - quartenary reactions (due to attributes) allow V3 to grasp nearby protomers on the viral spike -- these interactions lalow for neutralization sensitivity
    • After binding - coreceptor binding site forms, V3 move toward target cell membrane
      • V3 tip intiiated gp41 mediated fusion
    • By altering quartenary interactions, HIV can evade immune system detection and easily enter cells

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