Abstract
A promising strategy to neutralize HIV-1 is to target the gp41 spike subunit to block membrane fusion with the cell. We previously designed a series of single-chain proteins (named covNHR) that mimic the trimeric coiled-coil structure of the gp41 N-terminal heptad repeat (NHR) region and potently inhibit HIV-1 cell infection by avidly binding the complementary C-terminal heptad repeat (CHR) region. These proteins constitute excellent tools to understand the structural and thermodynamic features of this therapeutically important interaction. Gp41, as with many coiled-coil proteins, contains in core positions of the NHR trimer several highly conserved, buried polar residues, the role of which in gp41 structure and function is unclear. Here we produced three covNHR mutants by substituting each triad of polar residues for the canonical isoleucine. The mutants preserve their helical structure and show an extremely increased thermal stability. However, increased hydrophobicity enhances their self-association. Calorimetric analyses show a marked influence of mutations on the binding thermodynamics of CHR-derived peptides. The mutations do not affect however the in vitro HIV-1 inhibitory activity of the proteins. The results support a role of buried core polar residues in maintaining structural uniqueness and promoting an energetic coupling between conformational stability and NHR–CHR binding.
Highlights
HIV-1 infection remains a global pandemic with more than 38 million of people living with the virus and near 1.7 million new infections in 2019 [1]
All the covNHR core mutants were expressed in E. coli with good yields and showed high solubility, to the parent covNHR protein
We previously reported that the high affinity between the N-terminal heptad repeat (NHR) and C-terminal heptad repeat (CHR) regions is a result of cooperative contributions of several binding pockets along the NHR crevice
Summary
HIV-1 infection remains a global pandemic with more than 38 million of people living with the virus and near 1.7 million new infections in 2019 [1]. Active antiretroviral therapies (HAART) have improved considerably the life expectancy of infected patients, but the emergence of multidrug-resistant viral strains [3] and the appearance of adverse effects and drug–drug interactions in some patients [4] highlight the need for new improved antivirals and microbicides to combat the infection. Viral attachment to cells is initiated by gp120, which binds to the CD4 cell receptor and a co-receptor. This event triggers gp120 shedding from gp and a large conformational change in the latter that inserts its N-terminal fusion peptide in Biomolecules 2021, 11, 566.
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