Structural and functional properties of the membranotropic HIV-1 glycoprotein gp41 loop region are modulated by its intrinsic hydrophobic core

Abstract

The gp41 disulfide loop region switches from a soluble state to a membrane-bound state during the human immunodeficiency virus type 1 (HIV-1) envelope-mediated membrane fusion process. The loop possesses a hydrophobic core at the center of the region with an unusual basic residue (Lys-601). Furthermore, two loop core mutations, K601A and L602A, are found to inhibit HIV-1 infectivity while keeping wild type-like levels of the envelope, implying that they exert an inhibitory effect on gp41 during the membrane fusion event. Here, we investigated the mode of action of these mutations on the loop region. We show that the K601A mutation, but not the L602A mutation, abolished the binding of a loop-specific monoclonal antibody to a loop domain peptide. Additionally, the K601A, but not the L602A, impaired disulfide bond formation in the peptides. This was correlated with changes in the circular dichroism spectrum imposed by the K601A mutation. In the membrane, however, the L602A, but not the K601A, reduced the lipid mixing ability of the loop peptides, which was correlated with decreased α-helical content of the L602A mutant. The results suggest that the Lys-601 residue provides a moderate hydrophobicity level within the gp41 loop core that contributes to the proper structure and function of the loop inside and outside the membrane. Because basic residues are found between the loop Cys residues of several lentiviral fusion proteins, the findings may contribute to understanding the fusion mechanism of other viruses as well.

Keywords: Biophysics; HIV-1; Membrane Fusion; Membrane Proteins; Peptide Conformation; Peptide-Membrane Interaction; Viral Fusion Protein.

FIGURE 1.

Bioinformatics characterization and mutagenesis of the hydrophobic loop core. A, presentation of the loop structure devoid of the cysteine residues together with the six-helix bundle in the soluble hairpin conformation of the SIV fusion protein. This is the only available structure of the loop. The three-dimensional structure was taken from Caffrey et al. (13), PDB ID 1QCE. B, the location of the hydrophobic core within the loop region. The highest hydrophobic residues are blue, and the lowest hydrophobic residues are red. C, sequence homology in the loop hydrophobic core between HIV and SIV clades shows conservation of the basic residue (Lys or Arg) between the two Cys residues. D, alteration of the hydrophobic level of the gp41 loop core by mutagenesis analysis. Residue numbers correspond to the gp160 HIV-1 HXB2 variant.

FIGURE 2.

The hydrophobic core and its basic residue participate in the proper conformation of the loop as revealed by antibody binding. Binding capacity of loop-specific monoclonal antibodies to L27 WT loop peptides (□), L27 K601A mutant peptides (■), L27 L602A mutant peptides (▴), L42 WT loop peptides (○), and N27 control peptides (▵) is shown. A, analysis of T32 antibody binding. B, analysis of 240-D antibody binding. C, analysis of 246-D antibody binding. The amount of bound monoclonal antibodies was determined utilizing an ELISA protocol by monitoring the absorbance in 450 nm. Results are the mean ± S.D., n = 2 from a representative experiment out of two experiments. The fitting curves from the NLLSQ model that were used for calculating antibody binding constants are presented.

FIGURE 3.

The hydrophobic loop core and its basic residue contribute to disulfide bond formation. L27 peptides and their mutants were dissolved in PBS or in a lipid suspension of 100 μm large LUVs to give a concentration of 13.3 μm, and oxidation was monitored for several hours. A, the percentage of oxidation as determined by the difference in DTNB reaction with free thiol groups before and after the oxidation. Black columns represent oxidation in PBS, whereas white columns represent oxidation in the presence of LUVs. Results are the mean ± S.D. (n = 3) of the percentage of the oxidized fraction out of the total amount of the peptide. B, oxidation kinetics were monitored by injecting samples to the RP-HPLC at different time points for the L27 WT loop peptides (■), L27 K601A mutant peptides (▵), and L27 L602A mutant peptides (□). For each time point, the percentage of peptide oxidation (mean ± S.D., n = 3) was determined by calculating the amount of the oxidized peptide divided by the total amount of the peptide injected.

FIGURE 4.

Effect of the mutations on the secondary structure of the loop region utilizing CD spectroscopy. L27 WT peptides (black line), L27 K601A mutant peptides (▵), and L27 L602A mutant peptides (▴) were scanned at a concentration of 10 μm in solution (HEPES 5 mm, pH 7.4) (A) and in a membrane mimetic environment of 1% lysophosphatidylcholine in HEPES (B).

FIGURE 5.

Alterations in the hydrophobic core affect the binding of the loop region to zwitterionic membranes. WT L27 peptides (■), L27 K601A mutant peptides (▵), and L27 L602A mutant peptides (□) were titrated with increasing concentrations of PC:Chol (9:1) LUVs, and changes in fluorescence anisotropy (arbitrary units (au)) of their intrinsic Trp were measured. The fitting curve from the NLLSQ model is presented (Equation 1 under “Experimental Procedures”) that gives the membrane binding affinity constant.

FIGURE 6.

Full lipid mixing mediated by the loop region is modulated by its hydrophobic core. The loop peptide and its mutants were added to PC:Chol (9:1) LUVs in PBS containing 10% of pre-labeled LUVs (0.6 mol % NBD-PE and Rho-PE) and 90% of unlabeled LUVs. The peptide to lipid molar ratio was 0.05. The increase in NBD fluorescence intensity was measured until a plateau was reached, which represents full lipid mixing capacity and was normalized to the membrane-bound fraction of the peptides. Next, DTH (32 mm) was added to detect the inner leaflet mixing. A, a representative experiment for the loop peptides. B, a representative experiment for the N27 control peptides. C, mean ± S.D. (n = 3) of the percentage of the total lipid mixing of the L27 K601A mutant, L27 L602A mutant, and N27 peptides that was normalized to the WT L27. D, mean ± S.D. (n = 3) of the percentage of inner leaflet mixing out of the total lipid mixing for each of the peptides. au, arbitrary units.