Core Structure of gp41 from the HIV Envelope Glycoprotein

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Core Structure of gp41 from the HIV Envelope
Glycoprotein
David C. Chan1, 2, Deborah Fass1, 2, James M.
Berger1 and Peter S. Kim1, 2,  1 Whitehead
Institute for Biomedical Research, Cambridge,
Massachusetts 02142, USA?2 Department of Biology,
Howard Hughes Medical Institute, Massachusetts
Institute of Technology, Cambridge, Massachusetts
02142, USA
Presentation by Andrew Bowen
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Acquired Immunity

• Every nucleated cell in the body has MHC (Major
  Histocompatibility Complex 1) . Some also have
  MHC 2.
• T cells will be activated upon binding to MHC
• MHC 2 binds to helper T cells

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CD4 glycoprotein helps hold MHC 2 in place
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Human Immunodeficiency Virus
retrovirus (enveloped lentivirus) positive
strands, ssRNA genome genome copied into DNA
and integrated into the host cell genome
believed to have evolved from monkey viruses that
recombined
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Infection

• gp 160 a precursor protein is activated by
  cleavage.
• Becomes gp120 and gp41 on the virion surface
  attach to CD4 and the co- receptors CCR5 or
  CXCR4
• Infection of CD4 T cells depletes them from
  the circulation
• viral lysis of infected cells
• increased apoptosis
• killing by CD8 T cells

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gp120 and gp41 mechanism

• gp120 binds to CD4 and disassociates from gp41
• It is believed that this disassociation causes a
  conformational change in gp41 that is crucial for
  membrane fusion
• gp41 then fuses the two membranes together

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Why does it matter?

• The fusion of gp41 is very similar to other viral
  membrane-fusion proteins
• One big example Influenza virus
• Influenza has HA1 and HA2
• Fusion peptide has a high a-helical propensity
  and a 4-3 heptad repeat of hydrophobic residues,
  characteristic of coiled coils

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A coiled coil is a structural motif in proteins,
in which 2-71 alpha-helices are coiled together
like the strands of a rope
Coiled-coil motif (4-3 hydrophobic repeat) is a
heptad repeat of amino acids from a to g so that
a and d are hydrophobic and e and g predominantly
polar.
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• Understanding the structure of gp41 will give
  knowledge of how the protein functions
• Will open new fields of research in new drugs
  that may be able to inhibit membrane fusion
• Allow for a better understanding of virus
  membrane-fusion

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Complications

• It has not yet been possible to obtain a detailed
  structure for gp41, either alone or in complex
  with gp120.
• Therefore had to apply a protein-dissection
  approach
• Chose the regions N51 and C43
• The N51 peptide corresponds to the 4-3
  hydrophobic.
• repeat region adjacent to the fusion peptide,
  while the
• C43 peptide is derived from the region prior to
  the trans-membrane segment
• Unfortunately the N51/C43 is not
  thermodynamically favorable and will undergo
  permanent denaturation
• Instead used N36/C34

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X-Ray Crystallography

• Peptides were synthesized using
  9-fluorenylmethyloxycarbonyl (Fmoc) solid phase
  synthesis
• Crystals of N36/C34 were grown by sitting drop
  vapor diffusion
• Due to phase problem used the Heavy metal method
• Used Osmium in OsO4 form
• Crystal were hexagonal prisms belong to space
  group P321
• Measurements diffracted at four different
  wavelengths
• Electron density potential map at 2.0 Å

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The N36/C34 Complex

• Complex consists of a trimeric complex of
  heterodimers
• 35Å in diameter, 55Å in height
• Characterisic knobs into holes packing of N36
  at the a and d layers. Pack into cavities between
  four residues of adjacent helix
• Noncovalent bonding interactions of C34 into the
  grooves of N36

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Hydrophobic/philic trends

• N36 has heptad repeat
• Surface of coiled trimer is uncharged
• Grooves in trimer are extremely hydrophobic
• Surface of C34 is very charged
• Acidic and basic

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Trimer or Tetramer?

• Previous studies have suggested that the N34
  peptides form a tetramer verus trimer
• This is debunked in this experiment
• Nature of previous experiments without the C34
  will cause this to happen
• Reinforces importance of C34 to integrity of
  structure

3 or 4?
HYDROPHOBICITY IS KEY
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Similarities to SIV

• a and d residues of C43 pack against e and g
  residues of N36
• No non-conservative changes of e and g between
  SIV and HIV. Only 2 between a and d
• 13 non-conservative changes on N36 at f, b, and
  c
• 15 non-conservative changes on C34 at positions
  other than a and d
• Highly conserved hydrophobic regions. Shows the
  importance of the hydrophobicity (because its
  KEY)

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Looking deeper into the groove

• 16Å long, 7Å wide, and 5-6Å deep
• Four hydrophobic residues from C34 fit in here
  Ile-635, Trp-631, Asp-632 and Trp-628
• Left side of cavity Leu-566, Val-570, Lys-574,
  Gln-577
• Right side of cavity Leu-565, Leu-568, Trp-571,
  and Gly-572
• Floor of cavity Thr-569, Ile-573, and Leu-576
• Salt bridge with Lys-574 and Asp-632

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Similarities to Influenza and Mo-MLV

• The N36/C34 complex shows striking structural
  similarity to the low-pH-induced conformation of
  the influenza HA2 subunit (TBHA2) and to the TM
  subunit of Mo-MLV, each of which has been
  proposed to be a fusogenic conformation.
• Remarkably, the core of each of the three
  structures contains a three-stranded coiled coil
  that would be adjacent to the amino-terminal
  fusion peptide

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What state is N36/C34 in?

• Native or fusogenic state or both?
• 5 Reasons for it being the fusogenic state
• 1) N36/C34 folds in the absence of gp120. Similar
  to Influenza virus
• 2) Isolated gp41 core is exceedingly stable
  unlike the glycoprotein native counterpart
• 3) Mutations in gp41 that abolish infectivity and
  membrane fusion often map to residues that are
  expected to stabilize the gp41 core structure.
  Furthermore proves that interactions between N36
  and C34 are critical for membrane fusion. HIV was
  unable to form syncytia with cells.
• 4) This postulate is consistent with a large body
  of data on inhibition of HIV-1 infection by
  derivatives of peptides that make up the core
• 5) Similarities to Influenza and Mo-MLV

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Inhibitors of HIV

• Synthetic peptides containing approximately 40
  residues from gp41 that overlap, or include all
  of, the residues in N36 or C34 can be effective
  inhibitors, at micromolar to nanomolar
  concentrations, of HIV and syncytia formation
• N51 and C43 affect in negative-dominant manner
• C-peptide derivatives act by inhibiting coiled
  coil trimer from binding to viral gp41
• N-peptides derivatives act by inhibiting
  formation of coiled coil trimer within viral gp41

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Possible New Areas of Research

• The crucial highly conserved cavities of the
  N-peptide coiled trimer are attractive targets
  for new drugs
• New peptidomimetic or small molecule inhibitors
• Cavity covered earlier is particular point of
  study for new drugs
• It is also likely that other distinctive surface
  features exist in the interface of N and C
  helices of N51 and C43
• Current drugs do not attack HIV envelope

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Summary

• The similarities between Influenza, SIV, and
  Mo-MLV is undeniable
• It appears that these diverse viruses present
  fusion peptides to target cells via a common
  scaffold, in which the fusion peptides are atop a
  central, three-stranded coiled coil that is
  supported by additional, carboxy-terminal
  structures.
• Common structural features suggest that the rich
  body of work investigating the mechanism of
  membrane fusion for many other viruses, including
  influenza, is relevant for understanding the
  mechanism of HIV mediated membrane fusion
• Questions to still be answered, is coiled coil a
  spring loading mechanism? Is it also the native
  state? Does gp120 just expose this region once it
  disassociates?
• This gp41 core structure serves as the starting
  point for addressing essential questions like
  these about the mechanism of HIV entry into cells.

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HIV in 3D -Science and the National Science
Foundation, the annual competition awards entries
that "engage people worldwide and convey science
close up in novel and visually stimulating ways,"
according to a statement. Judging criteria
include visual impact, effective communication,
freshness, and originality. A Russian team led by
Ivan Konstantinov analyzed data from more than a
hundred scientific journals to digitally depict
HIV as close to the real thing as possible. The
two-tone color scheme shows HIV (orange)
attacking and fusing with an immune cell (gray).
The triangular cut-away shows how the virus
integrates itself to turn the cell into a virus
factory. (Get the facts on AIDS.)"We consider
such 3-D models as a new way to present and
promote scientific data about ubiquitous human
viruses," Konstantinov, of the Visual Science
Company, said in a statement.