Zrusso Biol 368 week 7

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Journal Club Prep

BIOL368/F11:Week 7

Biological Terms

  1. Oligomeric - a polymer molecule consisting of a small number of monomers. Retrieved from [1] on 10/12/11
  2. Chemokine - any of a group of chemotactic cytokines that are produced by various cells (as at sites of inflammation), that are thought to provide directional cues for the movement of white blood cells (as T cells, monocytes, and neutrophils), and that include some playing a role in HIV infection because the cell surface receptors to which they bind are also used by specific strains of HIV for entry into cells. Retrieved from [2] on 10/12/11
  3. Fusogenic - Facilitating fusion, especially relating to cells. Retrieved from [3] on 10/12/11
  4. Prophylactic - defending or protecting from disease or infection, as a drug. Retrieved from [4] on 10/12/11
  5. Ternary - consisting of three different elements or groups. Retrieved from [5] on 10/12/11
  6. Prolate - elongated along the polar diameter, as a spheroid generated by the revolution of an ellipse about its longer axis ( opposed to oblate). Retrieved from [6] on 10/12/11
  7. Interfacial - included between two faces. Retrieved from [7] on 10/12/11
  8. Antigenic - having the properties of any substance that can stimulate the production of antibodies and combine specifically with them. Retrieved from [8] on 10/12/11
  9. Proteolytic - the breaking down of proteins into simpler compounds, as in digestion. Retrieved from [9] on 10/12/11
  10. Glycocalyx - a polysaccharide or glycoprotein covering on a cell surface. Retrieved from [10] on 10/12/11

Paper Outline

  • Introduction
    • HIV-1, HIV-2 and their cousin the Simian immunodeficiency viruses (SIV) destroy CD4 lymphocytes in their hosts, which results in AIDS
    • Entry of HIV virus into host cells is mediated by viral envelope glycoproteins
      • These glycoproteins are arranged in oligomeric, most likely trimeric spikes along the surface of the virion
      • These spikes are anchored to the viral membrane by gp41 transmembrane protein
      • The surface of the spike is primarily gp120
    • gp120 contains five variable regions (V1-V5)
    • both conserved and variable gp120 regions are heavily glycosylated
      • this glycosylation probably modulates the immunogenicity and antigenicity of gp120
    • gp120 is the main target for antibodies
    • gp120 will bind to glycoprotein on CD4 and acts as main receptor
      • gp120 binds to the most amino-terminal of the four immunoglobulin like domains of CD4
      • mutagenesis has found critical regions in both gp120 and CD4 for binding
    • CD4 binding induces a conformation change in gp120 which exposes/forms a chemokine receptor
      • This chemokine receptor for CCR5 and CXCR4 serve as obligate secondary receptors for HIV entry into the cell
      • V3 is the principle determinant of chemokine receptor specificity
      • There are other more conserved regions of gp120 that seem to be involved in chemokine-receptor binding
      • CD4i (CD4 induced) antibodies block the binding of the gp120-CD4 complex to the chemokine receptor
    • HIV and related retroviruses belong to a class of enveloped fusogenic viruses, all which require post-translational cleavage for activation.
      • Some share protein sequence similarity while others are quite distinctive.
    • since gp120 is so important in receptor binding and in interactions with antibodies, info about it is important
      • In this paper is reported the crystal structure at 2.5 Å detail a partially deglycosylated HIV-1 gp120 core bound to a two domain fragment of CD4 receptor and to an antigen binding fragment (Fab) 17b, which acts on a CD4i epitope.
  • Structure Determination
    • Due to the fact that gp120 is extensively glycosylated and shows great conformational heterogeneity, radical modification of the protein surface was devised to image it.
      • truncations were made at the termini and at variable loops in various combinations from various strains. These variants were then heavily deglycosylated and produced complexes with
      • This was done because a theoretical analysis showed an increase in probable crystal formation with the reduction of surface heterogeneity and trials with multiple variants.
      • After many combinations, crystals were obtained of a ternary complex that contained a truncated gp120, the N-terminal of two domains of CD4, and a Fab from 17b.
    • the gp120 crystallized was from HIV-1 strain HXBc2
      • deletions of 52 residues from N-terminus and 19 from C-terminus.
      • Gly-Ala-Gly tripeptide substitutions for 67 V1/V2 loop residues and 32 V3 loop residues
      • removal of all sugar groups beyond the linkages between the two core N-acetyl-glucosamine residues.
      • removal of 90% of total carbohydrate but retains 80% of non-variable loop protein
      • capacity to interact with CD4 and relevant antibodies is preserved at or near wild-type levels.
  • Structure of gp120
    • deglycosylated core of gp120 approximately looks like a prolate ellipsoid, though the outline is heart-shaped.
    • core is made up of 25 β-sheets, 5 α-helices, and 10 defined loop segments
    • the polypeptide chain is folded into two main domains along with some digressions from this body
      • Inner domain (with respect to N and C termini) contains a two-helix, two-sheet bundle with a small five sheet β-sandwich at its termini-proximal end and a projection from the distal side where the V1/V2 stem originates.
      • Outer domain is a stacked double barrel that lies alongside inner domain so that the both barrel axes are roughly parallel to each other.
      • There is a ‘minidomain’ which is comprised of four antiparallel β-sheets that create a ‘bridging sheet’ that is in contact with both the inner and outer domains
    • the structure of gp120 has no precedent
      • domain-level searches revealed no similar structures, though missing terminal segments might account for this
      • there is fragmentary similarity for portions of outer domain but no sequence evidence to support an evolution argument.
    • This structure of core gp120 should be a prototype for the class
      • structure based alignment shows conservation despite the variability in HIV strains
        • HIV-2 is 35% similar
        • 77% and 51% for HIV-1 clade C and O respectively
        • Outer domain is more conserved with 86%, 72%, and 45% for HIV-1 C, HIV-1 O, and HIV-2 respectively
  • CD4-gp120 Interaction
    • CD4 is bound in a depression formed at the interface of the outer and inner domain along with the bridging sheet of gp120
    • the interaction buries a total of 742 Å2 from CD4 and 802 Å2 from gp120
    • the surface area actually in contact is much smaller than this due to mismatch in surface topography that creates large cavitites
    • there is a general complementation of electrostatic potential at the surfaces of contact, though not perfect
    • Phe43 and Arg59 of CD4 make multiple contacts with gp120
      • Phe43 accounts for 23% of all interatomic contact
    • several gp120 residues that are covered by CD4 are variable
      • this variation is due to the fact that they are in contact with a large water filled cavity
    • there are two cavities that are unusually large
      • larger cavity is lined with hydrophilic residues from both gp120 and CD4
        • while formally a cavity in the crystal structure, minor changes in side-chain orientation would make it accessible to solvents
        • residues lining the cavity are variable, but those directly surrounding this variable patch are conserved and their substitution affect CD4 binding
        • this cavity acts as a water buffer between gp120 and CD4 and is a variation island or ‘anti-hotspot’ located between conserved regions for CD4 binding, therefore might help virus escape from antibodies directed at its CD4 binding site.
      • the Phe43 cavity is very different, and is roughly spherical
        • located just beyond Phe43 and at the intersection of inner and outer domains and binding sheet
        • deeply buried and extends into interior of gp120
        • only a few water molecules are seen in this cavity and at the center of it is a large spherical density over 4 Å from any protein atom
        • its density does not match any major crystallization component and is currently unidentified.
        • residues that line the cavity are hydrophobic and are as highly conserved as the buried hydrophobic core of gp120
        • there is no steric hindrance but no substitutions to larger residues were found
        • such conservation indicates functional significance
        • many of the residues that line the Phe43 cavity interact with elements of the chemokine-receptor-binding region
    • the missing structures could not conceivably have a role in filling these cavities
  • Antibody Interface
    • 17b is a broadly neutralizing antibody isolated from an HIV-infected individual
      • Binds to a CD4i gp120 epitope that overlaps the chemokine receptor binding site
    • relative to other antibody-antigen pairs, has a small interface
    • 17b contact surface is very acidic, with hydrophobic contacts at the center
    • 17b epitope lies across base of bridging sheet, suggesting its integrity is needed for antigen binding
    • 17b binds on opposite side of CD4, suggesting that it stabilizes that bridge, allowing 17b to bind
    • great conservation among residues in contact with 17b
    • Note that in intact gp120, V2 and V3 loops may need to be bypassed for access to the conserved structures in the binding sheet and the epitope may further be protected by a CD4 induced conformation change.
  • Chemokine Receptor Site
    • overlaps with 17b binding site and also contains highly conserved residues
  • Oligomer & gp41 interaction
    • gp120 probably exists as a trimer complex with gp41 on the viron surface
      • the large electroneutral surface on the inner domain is probable site for this packing
    • N and C terminal regions are most important in binding to gp41
    • similar arrangement is seen in other viruses
  • Conformational Change in core of gp120
    • lots of evidence to suggest CD4 induces conformation change in gp120
      • Phe43 cavity would present a structural dilemma with the absence of CD4
      • 17b will bind to gp120, but at much lower affinity, until CD4 does
    • Phe43 seems to serve as linchpin without which the structure might collapse
    • It is believed that CD4 sees gp120 at an equilibrium of conformation states and creates a complex states and then inserts Phe43 to stabilize it.
  • Viral Evasion of Immune Responses
    • Analysis of antigenic structure of gp120 shows that most of protein surface is hidden by glycosylation
    • only two free surfaces
      • CD4 binding site which is shielded by V1 and V2 loops
      • chemokine receptor binding site which is shielded by V2 and V3 loops
  • Mechanistic implications for virus entry
    • HIV surface proteins fuse viral membrane to target cell membrane and gp120 is crucial part
    • gp120 functions as a positioning agent, both in finding a suitable target as well as lining up the viral spike
    • entry process initiated by binding of HIV to CD4
    • flexibility in CD4 orients chemokine receptor site towards cell membrane as well as exposes it
    • chemokine receptor triggers another conformation change which reveals gp41 ectodomain and initiates viral spike

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Assignment Pages

BIOL368/F11:Week 7

BIOL368/F11:Week 8

BIOL368/F11:Week 9

BIOL368/F11:Week 10

BIOL368/F11:Week 11

BIOL368/F11:Week 12

BIOL368/F11:Week 14