Conserved Residue Lys574 in the Cavity of HIV-1 Gp41 Coiled-coil Domain Is Critical for Six-helix Bundle Stability and Virus Entry

Abstract

The fusion-active HIV-1 gp41 core structure is a stable sixhelix bundle (6-HB) formed by its N-and C-terminal heptadrepeat sequences (NHR and CHR). A highly conserved, deep hydrophobic cavity on the surface of the N-helical trimer is important for stability of the 6-HB and serves as an ideal target for developing anti-human immunodeficiency virus (HIV) fusion inhibitors. We have recently identified several small molecule HIV-1 fusion inhibitors that bind to the gp41 cavity through hydrophobic and ionic interactions and block the gp41 6-HB formation. Molecular docking analysis reveals that these small molecules fit inside the hydrophobic cavity and interact with positively charged residue Lys 574 to form a conserved salt bridge. In this study, the functionality of Lys 574 has been finely characterized by mutational analysis and biophysical approaches. We found that substitutions of Lys 574 with non-conserved residues (K574D, K574E, and K574V) could completely abolish virus infectivity. With a set of wildtype and mutant N36 peptides derived from the NHR sequence as a model, we demonstrated that non-conservative Lys 574 substitutions severely impaired the stability and conformation of 6-HBs as detected by circular dichroism spectroscopy, native polyacrylamide gel electrophoresis, and enzyme-linked immunosorbent assay. The binding affinity of N36 mutants bearing non-conservative Lys 574 substitutions to the peptide C34 derived from the CHR sequence dramatically decreased as measured by isothermal titration calorimetry. These substitutions also significantly reduced the potency of N-peptides to inhibit HIV-1 infection. Collectively, these data suggest that conserved Lys 574 plays a critical role in 6-HB formation and HIV-1 infectivity, and may serve as an important target for designing anti-HIV drugs. Entry of human immunodeficiency virus type 1 (HIV-1)3 into target cells is mediated by its envelope glycoprotein (Env), a type I transmembrane protein consisting of surface subunit gp120 and non-covalently associated transmembrane subunit gp41 (1). Sequential binding of HIV-1 gp120 to its cell receptor CD4 and a coreceptor (CCR5 or CXCR4) can trigger a series of conformational rearrangements in gp41 to mediate fusion between viral and cellular membranes (2-4). Structurally, the gp41 ectodomain contains the N-terminal heptad-repeat sequence (NHR) and C-terminal heptad-repeat sequence (CHR), which are adjacent to the fusion peptide and the transmembrane segment, respectively (Fig. 1A). Crystallographic analyses demonstrated that the NHR and CHR associate to form a stable six-helix bundle (6-HB), representing a fusionactive gp41 core structure, in which three N-helices form an interior, parallel coiled-coil trimer, whereas three C-helices pack in an oblique, antiparallel manner into the highly conserved, deep hydrophobic cavity on the surface of the N-helical trimer (Fig. 1B) ) penetrate into the cavity causing an extensive interaction with the hydrophobic residues in the cavity. Considerable evidence imply that interheli...

Single-chain protein mimetics of the N-terminal heptad-repeat region of gp41 with potential as anti–HIV-1 drugs

“…For the unprotected peptide W34L un , the enthalpy is smaller, but binding is also entropically favored. He et al (30) reported a binding affinity of N36 for the W34L peptide in the submicromolar range (K d = 300 nM). However, both the binding enthalpy and entropy values were much more negative than those reported here, likely because of the contribution of the folding and association of the NHR trimer.…”

The M-T Hook Structure Is Critical for Design of HIV-1 Fusion Inhibitors

“…HIV-1 Single-cycle Infection-HIV-1 pseudoviruses were generated as described previously (27,28). Briefly, 293T cells (5 ϫ 10 6 cells in 15 ml of growth medium in a T-75 culture flask) were cotransfected with 10 g of an Env-expressing plasmid and 20 g of a backbone plasmid pSG3…”

“…Isothermal Titration Calorimetry (ITC)-ITC assay was performed using a ITC 200 microcalorimeter instrument (MicroCal) as described previously (27). In brief, 1 mM N36 dissolved in double distilled H 2 O was injected into the chamber containing 100 M C34 or MT-C34.…”

Broad Antiviral Activity and Crystal Structure of HIV-1 Fusion Inhibitor Sifuvirtide

“…Inhibition of HIV-1 Single Cycle Infection-HIV-1 pseudoviruses were generated as described previously (27,28). Briefly, 293T cells (5 ϫ 10 6 cells in 15 ml of growth medium in a T-75 culture flask) were cotransfected with 10 g of an Env-expressing plasmid, and 20 g of a backbone plasmid pSG3…”

“…The mixture was added to TZM-bl cells (10 4 /well) and incubated at 37°C for 48 h. Luciferase activity was measured using luciferase assay reagents (Promega, Madison, WI) and a luminescence counter (Promega) according to the manufacturer's instructions. The percent inhibition by the peptides and 50% inhibitory concentration (IC 50 ) values were calculated as described previously (27,28).…”