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

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

CD4 glycoprotein helps hold MHC 2 in place

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

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

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

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 α-helical propensity and a 4-3 heptad repeat of hydrophobic residues, characteristic of coiled coils

A coiled coil is a structural motif in proteins, in which 2-7[1] 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.

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

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

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

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

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

3 or 4? Trimer or Tetramer? HYDROPHOBICITY IS KEY 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

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)

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

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

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

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

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

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.

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.