How Structure Correlates to Function for Membrane Associated HIV-1 gp41 Constructs Corresponding to the N-terminal Half of the Ectodomain

Sackett, Kelly; Shai, Yechiel

doi:10.1016/j.jmb.2003.07.008

Cited by 28 publications

(65 citation statements)

References 69 publications

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“…The structure and function of the HIV-1 gp41 molecule have been extensively studied (10,15,25,35,51,55,56,66,67). Considerable evidence suggests that during the fusion process gp41 likely exists in at least three conformations: (i) a metastable prefusogenic state, which is stabilized by interactions with gp120 subunit; (ii) a prehairpin intermediate, formed by insertion of the hydrophobic fusion peptide into the target cell membrane and concurrent folding of the N-terminal trimeric coiled coil; and (iii) the fusogenic trimer-of-hairpins form, in which two ␣-helical regions, the NHR (adjacent to the Nterminal fusion peptide) and the CHR (near the C-terminal transmembrane segment), associate to form a highly stable six-helix bundle, bringing the viral and cellular membranes into close apposition (6).…”

Section: Discussionmentioning

confidence: 99%

Conserved Salt Bridge between the N- and C-Terminal Heptad Repeat Regions of the Human Immunodeficiency Virus Type 1 gp41 Core Structure Is Critical for Virus Entry and Inhibition

Liu

et al. 2008

J Virol

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The fusogenic human immunodeficiency virus type 1 (HIV-1) gp41 core structure is a stable six-helix bundle formed by its N-and C-terminal heptad repeat sequences. Notably, the negatively charged residue Asp 632 located at the pocket-binding motif in the C-terminal heptad repeat interacts with the positively charged residue Lys 574 in the pocket formation region of the N-terminal heptad repeat to form a salt bridge. We previously demonstrated that the residue Lys 574 plays an essential role in six-helix bundle formation and virus infectivity and is a key determinant of the target for anti-HIV fusion inhibitors. In this study, the functionality of residue Asp 632 has been specifically characterized by mutational analysis and biophysical approaches. We show that Asp 632 substitutions with positively charged residues (D632K and D632R) or a hydrophobic residue (D632V) could completely abolish Env-mediated viral entry, while a protein with a conserved substitution (D632E) retained its activity. Similar to the Lys 574 mutations, nonconserved substitutions of Asp 632 also severely impaired the ␣-helicity, stability, and conformation of six-helix bundles as shown by N36 and C34 peptides as a model system. Furthermore, nonconserved substitutions of Asp 632 significantly reduced the potency of C34 to sequestrate six-helix bundle formation and to inhibit HIV-1-mediated cell-cell fusion and infection, suggesting its importance for designing antiviral fusion inhibitors. Taken together, these data suggest that the salt bridge between the N-and C-terminal heptad repeat regions of the fusion-active HIV-1 gp41 core structure is critical for viral entry and inhibition.Infection with human immunodeficiency virus type 1 (HIV-1) is mediated by its envelope glycoprotein (Env), a type I transmembrane protein which is originally synthesized as the single, glycosylated, polyprotein precursor gp160 and subsequently cleaved by a cellular protease to yield gp120 and gp41 subunits (13,14,20,46,48). Upon binding of the HIV-1 Env surface subunit gp120 to the cell receptor CD4 and subsequently to a coreceptor (CCR5 or CXCR4), its transmembrane subunit gp41 is released to mediate fusion of viral and cellular membranes (20,25,54). Structurally, HIV-1 gp41 consists of extracellular, transmembrane, and cytoplasmic domains (Fig. 1A). Its extracellular domain (ectodomain) contains four major functional regions: a hydrophobic, glycine-rich fusion peptide; an N-terminal heptad repeat (NHR) (also called HR1), a C-terminal heptad repeat (CHR) (also called HR2), and a tryptophan-rich region. In the early 1990s, several peptides derived from the NHR (N peptides) and CHR (C peptides) were found to have potent anti-HIV activity (30,43,68,69). Although their mechanism of action was not known at that time, the unprecedented anti-HIV activity of these peptides opened a new avenue for developing antiviral drugs. A C peptide known as T20 (brand name, Fuzeon) has been successfully developed as a novel class of anti-HIV drugs for clinical use (36,37,50).The findin...

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Section: Discussionmentioning

confidence: 99%

Conserved Salt Bridge between the N- and C-Terminal Heptad Repeat Regions of the Human Immunodeficiency Virus Type 1 gp41 Core Structure Is Critical for Virus Entry and Inhibition

Liu

et al. 2008

J Virol

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“…8). Following the triggering of the envelope glycoproteins by receptors and coreceptors, the gp41 protein, which is free to oscillate, inserts the FP, most likely in the ␤-sheet conformation (39), into the target membrane, joining them together. gp41 monomers interact with both membranes through the FP, the PTM, and the loop; at the same time that this interaction is taking place, it might be capable of inducing the self-assembly of gp41 oligomers at the membrane surface where the fusion site will be located (20).…”

Section: Discussionmentioning

confidence: 99%

A Peptide Pertaining to the Loop Segment of Human Immunodeficiency Virus gp41 Binds and Interacts with Model Biomembranes: Implications for the Fusion Mechanism

Pascual

Moreno

Villalaı́n

2005

J Virol

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The human immunodeficiency virus gp41 envelope protein mediates the entry of the virus into the target cell by promoting membrane fusion. In order to gain new insights into the viral fusion mechanism, we studied a 35-residue peptide pertaining to the loop domain of gp41, both in solution and membrane bound, by using infrared and fluorescence spectroscopy. We show here that the peptide, which has a membrane-interacting surface, binds and interacts with phospholipid model membranes and tends to aggregate in the presence of a membranous medium and induce the leakage of vesicle contents. The results reported in this work, i.e., the destabilization and fusion of negatively charged model membranes, suggest an essential role of the loop domain in the membrane fusion process induced by gp41.

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“…In model systems, the NHR significantly boosts the fusogenic function of the FP, for consecutive FP/NHR fragments (24), in part by pre-organizing the FP region (29). The ␤-structure adopted by the FP in membranes, when stabilized by the NHR, is likely that which is required during viral fusion (30). FP/NHR constructs model the N-terminal half of the PHI conformation (target of commercially available fusion inhibitor, Fuzeon), provide information on functional synergy between distinctly different regions of gp41 (24), and may be useful for screening fusion inhibitors.…”

mentioning

confidence: 99%

Characterization of the HIV N-terminal Fusion Peptide-containing Region in Context of Key gp41 Fusion Conformations

Sackett

Wexler-Cohen

Shai

2006

Journal of Biological Chemistry

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Central to our understanding of human immunodeficiency virus-induced fusion is the high resolution structure of fragments of the gp41 fusion protein folded in a low energy core conformation. However, regions fundamental to fusion, like the fusion peptide (FP), have yet to be characterized in the context of the cognate protein regardless of its conformation. Based on conformation-specific monoclonal antibody recognition, we identified the polar region consecutive to the N36 fragment as a stabilizer of trimeric coiled-coil assembly, thereby enhancing inhibitory potency. This tertiary organization is retained in the context of the hydrophobic FP (N70 fragment). Our data indicate that the N70 fragment recapitulates the expected organization of this region in the viral fusion intermediate (N-terminal half of the pre-hairpin intermediate (N-PHI)), which happens to be the prime target for fusion inhibitors. Regarding the low energy conformation, we show for the first time core formation in the context of the FP (N70 core). The ␣-helical and coiled-coil stabilizing polar region confers substantial thermal stability to the core, whereas the hydrophobic FP does not add further stability. For the two key fusion conformations, N-PHI and N70 core, we find that the FP adopts a nonhelical structure and directs higher order assembly (assembly of coiled coils in N-PHI and assembly of bundles in the N70 core). This supra-molecular organization of coiled coils or folded cores is seen only in the context of the FP. This study is the first to characterize the FP region in the context of the folded core and provides a basic understanding of the role of the elusive FP for key gp41 fusion conformations.The crystal structure of the HIV 2 gp41 core, presented nearly a decade ago (1-3), served as the basis for the following: (i) models describing gp41 conformational changes during its fusogenic activity (4, 5); (ii) the molecular mechanism describing the ability of exogenous gp41 N-and C-helix fragments to arrest fusion (6, 7); and (iii) design of next generation gp41-derived fusion inhibitors targeting both N-terminal heptad repeat (NHR) and C-terminal heptad repeat (CHR) viral targets (8 -10). Despite extensive efforts to characterize gp41, high resolution structures lack the N-terminal and membraneactive fusion peptide (FP) region, and our understanding of this fundamentally essential region is likewise limited.Prior to fusion, gp41 is held in a metastable state by the surface subunit gp120, and the complex is organized as a trimer visualized as spikes on the virion surface (11). Following receptors binding by gp120, gp41 is freed from metastable constraints (12) and adopts an extended conformation (pre-hairpin intermediate (PHI)) characterized by exposed NHR and CHR regions (8, 13). Collapse of the PHI into a thermostable six-helix bundle (SHB) or core conformation, formed by packing of CHR helices in an anti-parallel manner into hydrophobic grooves of the NHR trimeric parallel coiled coil (1), is believed to draw together viral and c...

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How Structure Correlates to Function for Membrane Associated HIV-1 gp41 Constructs Corresponding to the N-terminal Half of the Ectodomain

Cited by 28 publications

References 69 publications

Conserved Salt Bridge between the N- and C-Terminal Heptad Repeat Regions of the Human Immunodeficiency Virus Type 1 gp41 Core Structure Is Critical for Virus Entry and Inhibition

Conserved Salt Bridge between the N- and C-Terminal Heptad Repeat Regions of the Human Immunodeficiency Virus Type 1 gp41 Core Structure Is Critical for Virus Entry and Inhibition

A Peptide Pertaining to the Loop Segment of Human Immunodeficiency Virus gp41 Binds and Interacts with Model Biomembranes: Implications for the Fusion Mechanism

Characterization of the HIV N-terminal Fusion Peptide-containing Region in Context of Key gp41 Fusion Conformations

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