Biomedical subjects

Kelly Sackett

Publications and source records attributed to Kelly Sackett.

8 recordsLinked to original sources

PubMed2006

Functional and structural characterization of HIV-1 gp41 ectodomain regions in phospholipid membranes suggests that the fusion-active conformation is extended.

HIV-1 entry into its host cell involves a sequential interaction whereby gp41 is in direct contact with the plasma membrane. Understanding the effect of membrane composition on the fusion mechanism can shed light on the unsolved phases of this complex mechanism. Here, we studied N36, a peptide derived from the N-heptad-repeat (NHR) of the gp41 ectodomain, its six helix bundle (SHB) forming counterpart C34, together with the N-terminal 70-mer wild-type peptide (N70), and additional gp41 ectodomain-derived peptides in the presence of two membranes, modeling inner and outer leaflets of the plasma membrane. Information on the structure of these peptides, their affinity towards phospholipids and their ability to induce vesicle fusion was gathered by a variety of fluorescence, spectroscopic and microscopy methods. We found that N36, having strong affinity towards phospholipids, prominently shifts conformation from alpha-helix in an outer leaflet-like zwitterionic membrane to beta-sheet in a membrane mimicking the negatively charged inner leaflet environment, leading to pronounced fusion-activity. Real-time atomic force microscopy (AFM) was used to study the peptides' effect on the membrane morphology, revealing severe bilayer perturbation and extensive pore formation. We also found, that the N36/C34 core is destabilized by electronegative, but not zwitterionic phospholipids. Taken together, our data suggest that the fusion-active pore forming conformation of gp41 is extended, upstream of the SHB. In this manner, folding of the ectodomain into a SHB might also serve as a negative regulator of fusion by impeding gp41 fusion-active surfaces, thus preventing irreversible damage to the cell membrane. This assumption is supported by the finding that pre-incubation of large unilamellar vesicles (LUV) with C-heptad repeat (CHR)-derived fusion inhibitors reduces the fusogenic activity of N-terminal peptides in a dose-dependant manner, and suggests that CHR-derived fusion inhibitors inhibit HIV entry in an analogous mechanism.

Cell Membrane↗

PubMed2006

Characterization of the HIV N-terminal fusion peptide-containing region in context of key gp41 fusion conformations.

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 alpha-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.

Antibodies, Monoclonal↗

PubMed2005

The HIV fusion peptide adopts intermolecular parallel beta-sheet structure in membranes when stabilized by the adjacent N-terminal heptad repeat: a 13C FTIR study.

The HIV gp41 protein mediates fusion with target host cells. The region primarily involved in directing fusion, the fusion peptide (FP), is poorly understood at the level of structure and function due to its toxic effect in expression systems. To overcome this, we used a synthetic approach to generate the N70 construct, whereby the FP is stabilized in context of the adjacent auto oligomerization domain. The amide I profile of unlabeled N70 in membranes reveals prominent alpha-helical contribution, along with significant beta-structure. By truncating the N terminus (FP region) of N70, beta-structure is eliminated, suggesting that the FP adopts a beta-structure in membranes. To assess this directly, (13)C Fourier-transformed infra-red analysis was carried out to map secondary structure of the 16 N-terminal hydrophobic residues of the fusion peptide (FP16). The (13)C isotope shifted absorbance of the FP was filtered from the global secondary structure of the 70 residue construct (N70). On the basis of the peak shift induced by the (13)C-labeled residues of FP16, we directly assign beta-sheet structure in ordered membranes. A differential labeling scheme in FP16 allows us to distinguish the type of beta-sheet structure as parallel. Dilution of each FP16-labeled N70 peptide, by mixing with unlabeled N70, shows directly that the FP16 beta-strand region self-assembles. We discuss our structural findings in the context of the prevailing gp41 fusion paradigm. Specifically, we address the role of the FP region in organizing supramolecular gp41 assembly, and we also discuss the mechanism by which exogenous, free FP constructs inhibit gp41-induced fusion.

Amino Acid Sequence↗

PubMed2005

The role of the N-terminal heptad repeat of HIV-1 in the actual lipid mixing step as revealed by its substitution with distant coiled coils.

The gp41 glycoprotein of HIV-1 is considered to be responsible for the actual fusion process between the virus and the host membranes. According to a prevailing model, gp41 trimer organization, directed by the N-terminal coiled-coil region (NHR), is essential for steps involved in the actual merging of viral and cellular membranes. This study addresses a major question: Is the specific sequence of the NHR obligatory for the fusion process, or can it be replaced by distant coiled coils that form different oligomeric states in solution? For this purpose we synthesized three known GCN4 coiled-coil mutants that oligomerize in solution into either dimers, trimers, or tetramers. These peptides were chemically ligated to the fusion peptide thereby creating three chimera peptides with different oligomeric tendencies in solution. These peptides were investigated, together with the 70-mer wild-type peptide (N70), regarding their structure in solution and membrane by using circular dichroism (CD) and FTIR spectroscopies, their ability to induce vesicle fusion, and their ability to bind phospholipid membranes by using surface plasmon resonance (SPR). Our results suggest that local assembly of fusion peptides, facilitated by coiled-coil oligomers, increases lipid mixing ability, probably by facilitating stronger binding of the fusion peptides to the opposing membrane as revealed by SPR. However, N70 is significantly more active than the other chimeras. Overall, the data indicate a correlation between the distinct conformation of N70 in solution and in membranes and its enhanced lipid mixing relative to the GCN4 chimeras.

Amino Acid Sequence↗

PubMed2004

The stability of the intact envelope glycoproteins is a major determinant of sensitivity of HIV/SIV to peptidic fusion inhibitors.

C-peptides derived from the HIV envelope glycoprotein transmembrane subunit gp41 C-terminal heptad repeat (C-HR) region are potent HIV fusion inhibitors. These peptides interact with the gp41 N-terminal heptad repeat (N-HR) region and block the gp41 six-helix bundle formation that is required for fusion. However, the parameters that govern this inhibition have yet to be elucidated. We address this issue by comparing the ability of C34, derived from HIV-1, HIV-2 and SIV gp41, to inhibit HIV-1, HIV-2 and SIV envelope-mediated fusion and the ability of these peptides to form stable six-helix bundles with N36 peptides derived from gp41 of these three viruses. The ability to form six-helix bundles was examined by circular dichroism spectroscopy, and HIV/SIV Env-mediated membrane fusion was monitored by a dye transfer assay. HIV-1 N36 formed stable helix bundles with HIV-1, HIV-2 and SIV C34, which all inhibited HIV-1 Env-mediated fusion at IC(50)<10nM. The three C34 peptides were poor inhibitors of HIV-2 and SIV fusion (IC(50)>100nM), although HIV-2 and SIV N36 formed stable helix bundles with SIV C34. Priming experiments with sCD4 indicate that, in contrast to HIV-1, HIV-2 and SIV Env do not expose their N-HR region to SIV C34 following CD4 binding, but rapidly proceed to co-receptor engagement and six-helix bundle formation resulting in fusion. Our results suggest that several factors, including six-helix bundle stability and the ability of CD4 to destabilize the envelope glycoprotein, serve as determinants of sensitivity to entry inhibitors.

Amino Acid Sequence↗

PubMed2004

The C- and the N-terminal regions of glycoprotein 41 ectodomain fuse membranes enriched and not enriched with cholesterol, respectively.

To infect target cells, HIV-1 employs a virally encoded transmembrane protein (gp41) to fuse its viral envelope with the target cell plasma membrane. We describe the gp41 ectodomain as comprised of N- and C-terminal subdomains, each containing a heptad repeat as well as a fusogenic region, whose organization is mirrored by the intervening loop region. Recent evidence indicates that the gp41 directed fusion reaction proceeds to initial pore formation prior to gp41 folding into its low energy hairpin conformation. This implies that exposed regions of the gp41 ectodomain are responsible for the bulk of the fusion work, probably through direct protein-membrane interactions. Prevalent fusion models contend that the gp41 ectodomain initially interacts with the target cell surface through its highly hydrophobic N terminus, which is believed to insert into the target membrane, thereby linking the virus to the target cell. This arrangement allows the N-terminal subdomain to interact with the target cell surface, whereas the C-terminal subdomain remains proximal to the virion, allowing interaction with the viral envelope. The composition of the viral envelope and the target cell surface differ due to the virus budding from raft microdomains. We show here that constructs corresponding to the C-terminal subdomain specifically destabilize ordered and cholesterol rich membranes (33 molar %), whereas the N-terminal subdomain is more effective in fusing both unordered cholesterol-free membranes and those containing lower amounts of cholesterol (10 molar %). Moreover we show that, in the context of the C-terminal subdomain, the heptad repeat contributes helical structure, which may describe the enhanced inhibitory effect of the C-terminal subdomain relative to the C-terminal heptad repeat (C34) alone. Our results are discussed in light of recent findings that showcase the role of exposed gp41 regions in effecting membrane fusion.

Amino Acid Sequence↗

PubMed2003

How structure correlates to function for membrane associated HIV-1 gp41 constructs corresponding to the N-terminal half of the ectodomain.

To address the structure-function relationship of discrete regions within the gp41 ectodomain, 70-residue peptide constructs corresponding to the N-terminal subdomain of the HIV-1 gp41 ectodomain were examined in a membrane-associated context. These fragments encompass both fusion peptide (FP) and N-terminal heptad repeat (NHR) regions, and model the N-terminal half of the pre-hairpin intermediate (PHI), which is believed to be the target of the potent entry inhibitor DP-178, recently approved by the FDA. Using mutants, we attempted to map the structural organization of the N-terminal subdomain. Our results suggest that the N-terminal subdomain contains two discrete structural regions: the FP adopts a beta-sheet conformation and the NHR is alpha-helical. This structural make-up is essential for fusogenic function, since loss of function mutants exhibit both a significant reduction in region-specific secondary structure as well as significant impairment in lipid mixing of large unilamellar vesicles. Our results, delineating membrane-associated structure of the FP region differ from previous ones by inclusion of the autonomous oligomerization domain (NHR), which likely contributes to stabilization of the FP structure. Correspondingly, the alpha-helical structure for the NHR, in context of the FP, correlates with structural predictions for this region in both the hairpin and PHI conformations during fusion. Based on our results, we postulate how oligomerization of regions in this sub-domain is essential for fusion pore formation.

Amino Acid Sequence↗

PubMed2002

The HIV-1 gp41 N-terminal heptad repeat plays an essential role in membrane fusion.

For many different enveloped viruses the crystal structure of the fusion protein core has been established. A striking conservation in the tertiary and quaternary arrangement of these core structures is repeatedly revealed among members of diverse families. It has been proposed that the primary role of the core involves structural rearrangements which facilitate apposition between viral and target cell membranes. Forming the internal trimeric coiled coil of the core, the N-terminal heptad repeat (NHR) of HIV-1 gp41 was suggested to have additional roles, due to its ability to bind biological membranes. The NHR is adjacent to the N-terminal hydrophobic fusion peptide (FP), which alone can fuse biological membranes. To investigate the role of the NHR in membrane fusion, we synthesized and functionally characterized HIV-1 gp41 peptides corresponding to the FP and NHR alone, as well as continuous peptides made of both FP and NHR (wild type and mutant). We show here that a consecutive, 70-residue peptide consisting of both the FP and NHR (gp41/1-70) has dramatic fusogenic properties. The effect of including the complete NHR, as compared to shorter 23-, 33-, or 52-residue N-terminal peptides, is illustrated by a leap in lipid mixing of phosphatidylcholine (PC) large unilamellar vesicles (LUV) and clearly delineates the synergistic role of the NHR in the fusion event. Furthermore, a mutation in the NHR that renders the virus noninfectious is reflected by a significant reduction in in vitro lipid mixing induced by the mutant, gp41/1-70 (I62D). Additional spectroscopic studies, characterizing membrane binding and apposition induced by the peptides, help to clarify the role of the NHR in membrane fusion.

Amino Acid Sequence↗