HIV gp41: conformational plasticity in response to cholesterol

Lai, A.L., Eswara Moorthy, A., Li, Y., Tamm, L.K. (2012) Fusion activity of HIV gp41 fusion domain is related to its secondary structure and depth of membrane insertion in a cholesterol dependent fashion.  Journal of Molecular Biology 418 (1-2): 3-15

Human immunodeficiency virus, HIV, attacks the human immune system by targeting CD4⁺ T- cells, which will be covered in PPS section 12.  The virus is coated in a lipid bilayer which supports trimeric glycoprotein complex gp120/gp41.  The gp41 trimer is transmembrane whilst the gp120 glycoprotein projects from the viral surface, recognising and binding the CD4 receptors and chemokine coreceptors expressed on the T-cell surface.  This binding provokes change in the interaction between the glycoproteins, causing gp41 to extend its N terminus towards the T-cell, leaving its C terminus firmly embedded in the viral lipid membrane.  The N terminus, which includes conserved hydrophobic residues, inserts into the lipid bilayer of the T-cell and then the elongated section between the two membranes refolds into a helical hairpin, so bringing the membranes close enough to fuse and allowing the virus to enter the cell.  Several therapies, for example Fuzeon®, target gp41 by inhibiting the formation of the six helix bundle (one hairpin per each of the three monomers).  This process is covered in section 7 of PPS under The Life Cycle of HIV.  

The mechanism of insertion of the gp41 fusion domain into the T-cell’s lipid bilayer is clearly of paramount importance in understanding the process of membrane fusion but the structure of the fusion domain has been controversial with several studies directly contradicting each other.  NMR studies of the gp41 fusion domain in solution in lipid micelles revealed an α helical structure but NMR studies of the fusion domain in solid state in lipid bilayers indicate a structure which is primarily composed of β sheets.  (NMR is a widely used spectroscopic technique which utilises the oscillations of nuclei in a strong magnetic field.  This technique is covered in the TSMB course but current PPS students will not be able to follow the link.)  Neither of the sets of conditions is close enough to physiological conditions to be conclusive and so a recent study, (Lai, A.L. et al., (2012)), was designed to examine the structure taken by the domain under a range of possible physiological conditions.

The cell membranes of both the virus and the T-cell contain approximately 30 mol% cholesterol.  (Mol% is mole percentage, the molar mass of a constituent as a percentage of the average molar mass of the sample.)  This cholesterol is not distributed evenly, however, but forms lipid rafts of high cholesterol concentration surrounded by cholesterol poor membrane.  This paper used circular dichroism (again, this link is to the TSMB course) to study the structure adopted by the gp41 fusion domain bound to a lipid bilayer in conditions of zero cholesterol, 20 mol% cholesterol and 30 mol% cholesterol.  Circular dichroism exploits the fact that different secondary structures absorb alternating circularly polarised light at characteristic wavelengths and so the percentage of α helical and β sheet secondary structure within a protein can be calculated.

The surprising results are that where there is an absence of cholesterol, the structure is clearly α helical.  As the cholesterol content increased, β sheet structures appear and α helical structures dissipate until at 30 mol% cholesterol the β sheets are seen to predominate.  Since the length of the domain in question is quite short it seems unlikely that it is a mixed structure but rather that the domain changes conformation as the lipid composition alters.  

To increase understanding of this, electron paramagnetic resonance, or EPR, was used.  The fusion domain was labelled at four points with a nitroxide which would give a saturation signal as it reacted with the oxygen that is more prevalent in the hydrophobic centre of the lipid bilayer.  This could be used to show that the α helical conformation penetrated the bilayer more deeply at approximately 8 Å while the β sheet was held slightly closer to the headgroup region.  This data could also be used to perform an innovative docking exercise.  Several structures of the fusion domain of gp41 in α helical conformation had been previously solved and the lowest energy one, PDB 2PJV, was selected and the four nitroxides were added to it computationally.  This structure was then introduced to a computational model of a lipid bilayer and rotations and translations were applied until the derived saturation signals gave the best fit with the experimentally measured ones.  The resulting model shows the α helix embedded beneath the phosphate headgroups with the hydrophobic sidechains extending into the hydrocarbon centre of the lipid bilayer.

Model of the fusion domain of HIV gp41 docking in the lipid bilayer

Lai, A.L., Eswara Moorthy, A., Li, Y., Tamm, L.K. (2012) Fusion activity of HIV gp41 fusion domain is related to its secondary structure and depth of membrane insertion in a cholesterol dependent fashion.  Journal of Molecular Biology 418 (1-2): 3-15

Surprisingly, it has been shown that membrane fusion between the virus and the T-cell is initiated whichever of the two conformations is adopted.   Given this, the question is raised as to the mechanistic implication of this extraordinary ability to switch conformations depending on local lipid conditions without altering functionality.  Does the fusion domain enter the target membrane at a cholesterol rich region in β sheet conformation or a cholesterol poor region in α helical conformation?  There is currently no answer but the study puts forward some appealing suggestions.

Possibly the insertion occurs across the boundary of the cholesterol rich raft.  In this case there may be a mix of conformations dependent on the exact location of each fusion domain.  Alternatively, the insertion may initiate in the lipid raft but then switch into the α helical conformation in order to pass through the less ordered cholesterol poor membrane.  This may be because this region of membrane is more conducive to fusion or because of the unproven hypothesis that the fusion domain interacts with the gp41 transmembrane domain to engender membrane fusion.  There are indications of this interaction in the fusion mechanism of the influenza virus and it is considered that a more deeply embedded α helix would provide a more attractive binding platform than a β sheet.  Having elucidated the structural effects of cholesterol on the fusion of the HIV virus with a human T-cell, the next step will be to understand the mechanistic implications as a route to possible new therapies.