Structural and Thermodynamic Analysis of HIV-1 Fusion Inhibition Using Small gp41 Mimetic Proteins

Abstract

Development of effective inhibitors of the fusion between HIV-1 and the host cell membrane mediated by gp41 continues to be a grand challenge due to an incomplete understanding of the molecular and mechanistic details of the fusion process. We previously developed singlechain, chimeric proteins (named covNHR) that accurately mimic the N-heptad repeat (NHR) region of gp41 in a highly stable coiled-coil conformation. These molecules bind strongly to peptides derived from the gp41 C-heptad repeat (CHR) and are potent and broad HIV-1 inhibitors. Here, we investigated two covNHR variants differing in two mutations, V10E and Q123R (equivalent to V38E and Q40R in gp41 sequence) that reproduce the effect of HIV-1 mutations associated to resistance to fusion inhibitors, such as T20 (enfuvirtide). A detailed calorimetric analysis of the binding between the covNHR proteins and CHR peptides (C34 and T20) reveals drastic changes in affinity due to the mutations as a result of local changes in interactions at the site of T20 resistance. The crystallographic structure of the covNHR:C34 complex shows a virtually identical CHR-NHR binding interface to that of the post-fusion structure of gp41 and underlines an important role of buried interfacial water molecules in binding affinity and in development of resistance against CHR peptides. Despite the great difference in affinity, both covNHR variants demonstrate strong inhibitory activity for a wide variety of HIV-1 strains. These properties support the high potential of these covNHR proteins as new potent HIV-1 inhibitors. Our results may guide future inhibition approaches.

Thermodynamic dissection of the interface between HIV-1 gp41 heptad repeats reveals cooperative interactions and allosteric effects

“…Recently, we have designed, produced and characterized several protein molecules that mimic the structure of the NHR helices trimer of the intermediate state of fusion of gp41 [25,26]. These proteins, called covNHR, consist of a single polypeptide chain with three helical regions that fold as a trimeric bundle with a structure highly similar to the NHR gp41 region.…”

“…They are also very stable and highly soluble. We have shown that covNHR molecules bind with high affinity to peptides derived from the CHR region of gp41 and neutralize HIV cell infection with high potency (IC 50 in the low nanomolar range) for a wide variety of HIV strains and even for T20-resistant variants [26].…”

“…In our most recent study, we have solved the crystallographic structure of one of these covNHR variants (covNHR-VQ) in complex with the CHR-derived peptide C34 [26].…”

“…The most prominent one is the deep hydrophobic pocket (HP) [1], which has been widely used as a target for the discovery of small-molecule inhibitors, short peptides and antibodies. Another important site of interaction occurs at an N-terminal pocket (NTP), where polar CHR and NHR residues establish a dense network of polar interactions and hydrogen bonds mediated by two interfacial water molecules [26].…”

“…Because our covNHR proteins are very well behaved and bind the CHR peptides with 1:1 stoichiometry, they are particularly amenable for thermodynamic studies of the CHR-NHR interaction. For this reason in this study, we employ the previously described covNHR proteins [26] to perform a detailed thermodynamic dissection of the NHR-CHR interaction. Using isothermal titration calorimetry (ITC) we studied the binding of several different length peptides encompassing every CHR binding motif.…”