Large Changes in the CRAC Segment of gp41 of HIV Do Not Destroy Fusion Activity if the Segment Interacts with Cholesterol

Abstract

The membrane-proximal external region (MPER) of the gp41 fusion protein of HIV is highly conserved among isolates of this virus and is considered a target for vaccine development. This region also appears to play a role in membrane fusion as well as localization of the virus to cholesterol-rich domains in membranes. The carboxyl terminus of MPER has the sequence LWYIK and appears to have an important role in cholesterol interactions. We have tested how amino acid substitutions that would affect the conformational flexibility of this segment could alter its interaction with cholesterol. We studied a family of peptides (all peptides as N-acetyl-peptide amides) with P, G or A substituting for W and I of LWYIK. The peptide having the largest effect on cholesterol distribution in membranes was the most flexible one, LGYGK. The corresponding mutation in gp41 resulted in a protein retaining 72% of the fusion activity of the wild type protein. Two other peptides were synthesized, also containing two Gly residues, GWGIK and LWGIG, did not have the ability to sequester cholesterol as efficiently as LGYGK did. Making the corresponding mutants of gp41 showed that these other two double Gly substitutions resulted in proteins that were much less fusogenic, although they were equally well expressed at the cell surface. The study demonstrates that drastic changes can be made in the LWYIK segment with the retention of a significant fraction of the fusogenic activity, as long as the mutant proteins interact with cholesterol.

Cholesterol Interaction with Proteins That Partition into Membrane Domains: An Overview

“…Thus, the L679I mutant has about the same fusogenic activity as does the W680G, I682G mutant. Nevertheless, the peptide LGYGK is much more effective in sequestering cholesterol (Vishwanathan et al, 2008b) than is the peptide IWYIK (Vishwanathan et al, 2008a). This supports the CRAC hypothesis for cholesterol interactions since LGYGK is a CRAC sequence but IWYIK is not.…”

“…I or V in the first position of the CRAC segment result in poor cholesterol sequestering activity. We therefore systematically substituted either G, P or A in positions 2 or 4 of LWYIK to replace W and I (Vishwanathan et al, 2008b). These are the variable positions of the CRAC sequence; hence all of these peptides were CRAC motifs.…”

Targeting HIV-1 gp41 in Close Proximity to the Membrane Using Antibody and Other Molecules

“…Relatively conserved mutations to the first, third, and fifth residues significantly weaken peptide binding to Chol, and the conserved mutant IWYIK shows no preferential binding to Chol [82,84,85,87]. The LWYIK peptide was found to reside slightly closer to the center of the bilayer than mutant peptides, and the wildtype sequence is predicted to form stronger bonds with the hydroxyl group of Chol than other sequences [82,84,87].…”

“…It has been shown that peptide LWYIK partitions into the membrane interface and induces formation of Chol-rich domains in mixtures of 1-stearoyl-2-oleoyl-PC (SOPC) with 40-50% Chol, which is similar to the Chol concentration in the viral membrane [82][83][84]. The LWYIK peptide also shifts from a surface-bound to a membrane-inserted state and pellets with lipids in the presence, but not absence, of Chol [85]. The peptide has both van der Waals interactions with hydrophobic surfaces of Chol and electrostatic interactions with its hydroxyl group [82,84].…”

Membrane-Transferring Regions of gp41 as Targets for HIV-1 Fusion Inhibition and Viral Neutralization

“…MPER carboxy-terminal LWYIK sequence has been identified as a potential "cholesterol recognition/interaction amino acid consensus" (CRAC) domain functional in fusion [83][84][85][86][87]. Recently reported experimental evidence and modeling studies provide the physicochemical grounds for a direct interaction of MPER-CRAC with Chol [84,85,88,89]. MPER-Chol interaction would be based on two capacities of CRAC sequence, namely, wrapping and blocking of the interfacial cholesterol OH group by H-bond interactions, and stacking of aromatic side chains with A ring of cholesterol.…”