Angela A. Garibaldi Week 7

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Structure of an HIV gp120 envelope glycoprotein in complex with the CD4 receptor and a neutralizing human anti-body

Introduction

  • HIV destroys CD4+ lymphocytes

Basic Structure

  • Viral envelope (responsible for entry of HIV to host cells)has glycoproteins which contain oligomeric spikes, trimeric spikes on the surface
    • gp41 (transmembrane envelope glycoprotein) anchors the spikes in the viral membrane
    • spike surface made of gp120 (exterior envelope glycoprotein) which have non-covalent interactions with each subunit of trimeric gp41 complex
  • 5 variable regions
    • 1-4 form loops exposed to the surface which are bonded at their bases by disulfide bonds.

Basic Entry and Binding

  • conserved regions of gp120 form discontinuous structures that are essential for interacting with the gp41 ectodomain, and target cell's viral receptors
  • gp120 glycoprotein is target for neutralizing antibodies elicited during normal infection
    • variable and conserved regions of gp120 highly glycosylated.
    • glycosylation and variability of gp120 important in antigen elicitation of an adaptive immune response and ability of a chemical structure "Antigen" to bind specifically with T cell receptors or Antibodies/B cell receptors of gp120 glycoprotein
  • immunodeficiency viruses enter primate host cells gp120 envelope glycoprotein binding to the CD4 glycoprotein (1ary receptor)
    • gp120 binds most amino-terminal domain of CD4 (1 of 4).
  • Important items for binding:
  1. CD4 structure similar/same to CDR2 region of immunoglobulins for gp120 binding
  2. conserved gp120 residues for CD4 binding
  • When CD4 binds it causes conformational changes in gp120, which expose or form binding sites for CCR5,CXCR4, which act as secondary receptors for viral entry.
    • exposures conserved gp120 structures also, detected by improved binding of gp120 antibodies which block binding of gp120-CD4 complexes to the chemokine receptor (referred to as CD4i, CD4-induced antibodies
    • CD4 causes conformational changes in envelope glycoproteins causing shedding of gp120 from complex
  • HIV and related retroviruses:
  1. require post-translational cleavage for activation
  2. transmembrane coat proteins (gp41 equiv) share similar sequences in N-terminal fusion peptides
  3. share common features of virus entry
  4. distinctive in modes of entry
  5. individualized receptors
  6. specialized exterior coat proteins (gp120 equiv)
  7. distinct mech. for receptor-mediated triggering of fusion

Structure

Structure determination

  • Crystallization by modifying protein surface
    • used gp120 from multiple different strains
  • Major steps utilized:
  1. deglosylation
  2. produce complexes using different ligands that would bind things together in different combinations
  3. screened ~20 variants/ligands to get crystals (ternary, truncated, N-terminal (2) domains (DID2) of CD4, Fab from human 17b.
  4. crystallized gp120 that was ultimately chosen from HXBc2 strain of HIV-1
  5. ternary structure solved by molecular replacement, isomorphous replacement, density modification

Structure of gp120

  • prolate ellipsoid 50x50x25 A , heart shaped
  • outer stacked double barrel that share hydrophobic core
  • (distal end/antiparallel 4 stranded) bridging sheet - excised V3, excursion via loop LF into β-hairpin, β20-β21 (H-bond with V1/V2 stem coming out of the inner domain)
  • This overall structure has nothing previous that it is similar too in structure.'
  1. no similarities when looking at inner domains
  2. missing terminal sections might prove this wrong in the future
  3. some similarities in outer domains to part of the promoter of FabA dehydrase, and dUTP pyrophosphatase (barrels).
  4. use as prototype due to considerable conservation rates (35%-51% depending on strain).
  5. Inner domain (of hydrophobic cores) more conserved than outer domain (Barrels)

Figure 1

  • side chain of Phe43 on CD4
  • N and C termini of gp120 are missing
  • gp41, viral membrane, located toward top of diagram
  • target membrane at base of diagram

Figure 2

  • 90 degree rotation of Fig 1 has viral membrane above, target membrane below, C-terminal tail of CD4 coming out of page
  • A and C' - spatial proximity between strands= main-chain H bonding
    • sequences with high variability in the loops are circled
  • B(topology diagram) represents orientation of a and c
  • C-stereo plot of an α-carbon trace
  • D is the structure based sequence alignment
    • 'Solvent accessibility noted by open/half-filled/filled circle system
    • Sequence variability noted by hash mark system
    • gp120 residues in "direct contact" with CD4 noted by an *, this way is stricter than the solvent accessible surface system

Interactions

CD4-gp120 interaction

Figure 3

Interfacial Cavities

Antibody interface

Figure 4

Oligomer and gp41 interactions

Conformational change in core gp120

Figure 5

Viral evasion of immune responses

Mechanistic implications for virus entry

Methods

Protein production, crystallization and data collection

Structure determination and refinement

Table 1

Structure Analysis

Definitions

  1. epitope -That part of an antigenic molecule to which the T-cell receptor responds; a site on a large molecule against which an antibody will be produced and to which it will bind. biology-online.org dictionary
  2. chemokine - class of pro-inflammatory cytokines that have the ability to attract and activate leukocytes. They can be divided into at least three structural branches: c (chemokines, c), cc (chemokines, cc), and cxc (chemokines, cxc), according to variations in a shared cysteine motif. biology-online.org dictionary Small secreted proteins that stimulate chemotaxis of leucocytes. Chemokines can be subdivided into classes on the basis of conserved cysteine residues. The a -chemokines (IL-8, NAP-2, Gro- a , Gro- g , ENA-78 and GCP-2) have conserved C-X-C motif and are mainly chemotactic for neutrophils; the b -chemokines (MCP-1-5, MIP-1 a , MIP-1 b , eotaxin, RANTES) have adjacent cysteines (C-C) and attract monocytes, eosinophils or basophils; the g -chemokines have only one cysteine pair and are chemotactic for lymphocytes (lymphotactin), the d -chemokines are structurally rather different being membrane-anchored, have a C-X-X-X-C motif and are restricted (so far) to brain (neurotactin). Human genes for the a -chemokines are on Chr 4 and 10, for b -chemokines on Chr 17, for lymphotactin on Chr 1 and for neurotactin on Chr16. The receptors are G-protein coupled.Dictionary of Cell and Molecular Biology
  3. oligomeric spike/complex -
  4. trimeric spike/complex -
  5. fusogenic - Facilitating fusion of the viral envelope with the cellular plasma membrane. Nature Review Glossary
  6. anti-hotspot -
  7. cavity - a hollow place or space or a potential space, within the body or in one of its organs, it may be normal or pathological. biology-online.org dictionary
  8. linchpin - 1.A locking pin inserted in the end of a shaft, as in an axle, to prevent a wheel from slipping off. 2. A central cohesive element Farlex Dictionary
  9. humoral immune response - Those immune responses mediated by antibody. biology-online.org dictionary
  10. oligomeric occlusion - 1. The act of closure or state of being closed. 2. The relationship between all of the components of the masticatory system in normal function, dysfunction and parafunction.3. Momentary complete closure of some area in the vocal tract, causing stoppage of the breath and accumulation of pressure. biology-online.org dictionary
  11. glycocalyx - The region, seen by electron microscopy, external to the outer dense line of the plasma membranethat appears to be rich in glycosidic compounds such as proteoglycans and glycoproteins. Since these molecules are often integral membrane proteins and may be denatured by the processes of fixation for electron microscopy, it might be better to avoid the term or to refer to membrane glycoproteins or to proteoglycans associated with the cell surface. Dictionary of Cell and Molecular Biology
  12. ectodomain- is the part of a membrane protein that extends into the extracellular space (the space outside a cell). Ectodomains are usually the part of a protein that initiate contact with surface which leads to signal transduction. In SARS-CoV the ectodomain of the spike protein is responsible for attachment to and entry into cells during infection. Wikipedia
  13. fab -
  14. α-carbon trace -
  15. molecular replacement (technique) -
  16. isomorphous replacement (tech) -
  17. density modification (tech)-


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