Internal Dynamics of the Homotrimeric HIV‐1 Viral Coat Protein gp41 on Multiple Time Scales

Lakomek, Nils‐Alexander; Kaufman, Joshua D.; Stahl, Stephen J.; Louis, John M.; Grishaev, Alexander; Wingfield, Paul T.; Bax, Ad

doi:10.1002/ange.201207266

Cited by 30 publications

(89 citation statements)

References 31 publications

(45 reference statements)

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“…Our SEC monomer result is consistent with the SEC of large ectodomain constructs done by other groups (Figure 1A from ref (48) and Figure S1 from ref (51)) although this monomer interpretation was typically not made by the authors of these papers. For these latter studies, the construct was NHR + native loop + CHR, FP + NHR + native loop + CHR + MPER + TM, or short NHR + short loop + short CHR.…”

Section: Discussionsupporting

confidence: 91%

Folded Monomers and Hexamers of the Ectodomain of the HIV gp41 Membrane Fusion Protein: Potential Roles in Fusion and Synergy Between the Fusion Peptide, Hairpin, and Membrane-Proximal External Region

Banerjee

Weliky

2014

Biochemistry

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HIV is an enveloped virus and fusion between the HIV and host cell membranes is catalyzed by the ectodomain of the HIV gp41 membrane protein. Both the N-terminal fusion peptide (FP) and C-terminal membrane-proximal external region (MPER) are critical for fusion and are postulated to bind to the host cell and HIV membranes, respectively. Prior to fusion, the gp41 on the virion is a trimer in noncovalent complex with larger gp120 subunits. The gp120 bind host cell receptors and move away or dissociate from gp41 which subsequently catalyzes fusion. In the present work, large gp41 ectodomain constructs were produced and biophysically and structurally characterized. One significant finding is observation of synergy between the FP, hairpin, and MPER in vesicle fusion. The ectodomain-induced fusion can be very efficient with only ∼15 gp41 per vesicle, which is comparable to the number of gp41 on a virion. Conditions are found with predominant monomer or hexamer but not trimer and these may be oligomeric states during fusion. Monomer gp41 ectodomain is hyperthermostable and has helical hairpin structure. A new HIV fusion model is presented where (1) hemifusion is catalyzed by folding of gp41 ectodomain monomers into hairpins and (2) subsequent fusion steps are catalyzed by assembly into a hexamer with FPs in an antiparallel β sheet. There is also significant interest in the gp41 MPER because it is the epitope of several broadly neutralizing antibodies. Two of these antibodies bind our gp41 ectodomain constructs and support investigation of the gp41 ectodomain as an immunogen in HIV vaccine development.

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Section: Discussionsupporting

confidence: 91%

Folded Monomers and Hexamers of the Ectodomain of the HIV gp41 Membrane Fusion Protein: Potential Roles in Fusion and Synergy Between the Fusion Peptide, Hairpin, and Membrane-Proximal External Region

Banerjee

Weliky

2014

Biochemistry

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“…Recently, we found that the CHR, MPER, and TM regions of a much longer gp41 construct (residues 512-705; see Fig. 1A) are subject to extensive conformational exchange processes in the presence of DPC (32). Although sedimentation equilibrium centrifugation and SEC-MALS data unambiguously showed that this gp41 remains trimeric under such conditions, the chemical shifts of the NHR residues in gp41 512-705 correlate much closer with the shifts of the Core S monomer (R 2 = 0.76 and 0.74 for the 1 H N and secondary 13 C α chemical shifts, respectively) than with the Core S trimer [R 2 = 0.24 (δ 1 H N ) and R 2 = 0.55 (Δδ 13 C α )] (Fig.…”

Section: Discussionmentioning

confidence: 98%

“…6C). Formation of these complexes then depends on a competition between intermolecular association of the NHR and CHR helices, including their FPPR and MPER extensions, and membrane binding of these lipophilic regions (32,34). Bundling of MPER with FPPR residues stabilizes the 6HB state (9) but only becomes kinetically accessible after these regions have progressed to a state of close spatial proximity (Fig.…”

Section: Discussionmentioning

confidence: 99%

Dissociation of the trimeric gp41 ectodomain at the lipid–water interface suggests an active role in HIV-1 Env-mediated membrane fusion

Roche

Louis

Grishaev

et al. 2014

Proc. Natl. Acad. Sci. U.S.A.

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The envelope glycoprotein gp41 mediates the process of membrane fusion that enables entry of the HIV-1 virus into the host cell. The actual fusion process involves a switch from a homotrimeric prehairpin intermediate conformation, consisting of parallel coiled-coil helices, to a postfusion state where the ectodomains are arranged as a trimer of helical hairpins, adopting a six-helix bundle (6HB) state. Here, we show by solution NMR spectroscopy that a water-soluble 6HB gp41 ectodomain binds to zwitterionic detergents that contain phosphocholine or phosphatidylcholine head groups and phospholipid vesicles that mimic T-cell membrane composition. Binding results in the dissociation of the 6HB and the formation of a monomeric state, where its two α-helices, N-terminal heptad repeat (NHR) and C-terminal heptad repeat (CHR), become embedded in the lipid-water interface of the virus and host cell. The atomic structure of the gp41 ectodomain monomer, based on NOE distance restraints and residual dipolar couplings, shows that the NHR and CHR helices remain mostly intact, but they completely lose interhelical contacts. The high affinity of the ectodomain helices for phospholipid surfaces suggests that unzippering of the prehairpin intermediate leads to a state where the NHR and CHR helices become embedded in the host cell and viral membranes, respectively, thereby providing a physical force for bringing these membranes into close juxtaposition before actual fusion.T he first step of HIV infection involves fusion of the viral and target cell membranes, a process mediated by the viral envelope glycoprotein Env, consisting of subunits gp120 and gp41 (1). The envelope proteins form a noncovalent complex on the viral surface with the trimerized gp41 transmembrane subunit sequestered by three gp120 surface subunits (2-5). Binding of gp120 to the cell surface receptors CD4 and chemokine receptors CXCR4 or CCR5 triggers a cascade of conformational changes that disrupt the interactions between gp41 and gp120 and result in an extended gp41 conformation (1, 6). In this extended prefusion state, the highly hydrophobic N-terminal fusion peptide (FP) of gp41 anchors in the host cell membrane, while being spatially remote from its transmembrane domain (TM), which traverses the viral membrane (7,8). After the host cell and viral membranes have fused, the gp41 ectodomain, which links the FP and TM domains, has transitioned into a C3-symmetric six-helix bundle (6HB), with the FP in physical proximity to the TM domain (9). The refolding of gp41 trimers into the highly stable 6HB arrangement is believed to overcome the large free-energy barrier of membrane fusion. Several atomic resolution structures of the 6HB postfusion state have been solved by X-ray crystallography, confirming that the C-terminal heptad repeat (CHR) helices pack in an antiparallel manner into the conserved hydrophobic grooves formed at the surface of the central trimer of N-terminal heptad repeat (NHR) helices (10-12).Contrary to the postfusion state, structural features...

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“…The ectodomain can also be hexameric or more highly aggregated and the oligomerization states depend on pH, protein sequence, and solution additives [7,8]. Protein monomers are also predominant at low pH for gp41 constructs that include the TM domain although it is not known whether or not the TM domains of the trimer dissociate during HIV/cell fusion [9]. …”

Section: Introductionmentioning

confidence: 99%

pH-dependent vesicle fusion induced by the ectodomain of the human immunodeficiency virus membrane fusion protein gp41: Two kinetically distinct processes and fully-membrane-associated gp41 with predominant β sheet fusion peptide conformation

Ratnayake

Sackett

Nethercott

et al. 2015

Biochimica et Biophysica Acta (BBA) - Biomembranes

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The gp41 protein of the Human Immunodeficiency Virus (HIV) catalyzes fusion between HIV and host cell membranes. The ~180-residue ectodomain of gp41 is outside the virion and is the most important gp41 region for membrane fusion. The ectodomain consists of an apolar fusion peptide (FP) region followed by N-heptad repeat (NHR), loop, and C-heptad repeat (CHR) regions. The FP is critical for fusion and is hypothesized to bind to the host cell membrane. Large ectodomain constructs either with or without the FP are highly aggregated at physiologic pH but soluble in the pH 3–4 range with hyperthermostable hairpin structure with antiparallel NHR and CHR helices. The present study focuses on the large gp41 ectodomain constructs “Hairpin” (HP) containing NHR+loop+CHR and “FP-Hairpin” (FP-HP) containing FP+NHR+loop+CHR. Both proteins induce rapid and extensive fusion of anionic vesicles at pH 4 where the protein is positively-charged but do not induce fusion at pH 7 where the protein is negatively charged. This observation, along with lack of fusion of neutral vesicles at either pH supports the significance of attractive protein/membrane electrostatics in fusion. The functional role of the hydrophobic FP is supported by increases in the rate and extent of fusion for FP-HP relative to HP. There are two kinetically distinct fusion processes at pH 4: (1) a faster ~100 ms−1 process with rate strongly positively correlated with vesicle charge; and (2) a slower ~5 ms−1 process with extent strongly inversely correlated with this charge. The faster charge-dependent process is likely related to the electrostatic energy released upon initial monomer protein binding to the vesicle. After dissipation of this energy, the subsequent slower process is likely due to the equilibrium membrane-associated structure of the protein. The slower process may be more physiologically relevant because HIV/host cell fusion occurs at physiologic pH with gp41 restricted to the narrow region between the two membranes. Previous solid-state NMR (SSNMR) of membrane-associated FP-HP has supported protein oligomers with FP’s in an intermolecular antiparallel β sheet. There was an additional population of molecules with α helical FPs and the samples likely contained a mixture of membrane-bound and -unbound protein. For the present study, samples were prepared with fully membrane-bound FP-HP and subsequent SSNMR showed dominant β FP conformation at both low and neutral pH. The fusogenic structure is likely β for the slow and perhaps the fast process. SSNMR also showed close contact of the FP with the lipid headgroups at both low and neutral pH whereas the NHR+CHR regions had contact at low pH and were more distant at neutral pH, consistent with the protein/membrane electrostatics.

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Internal Dynamics of the Homotrimeric HIV‐1 Viral Coat Protein gp41 on Multiple Time Scales

Cited by 30 publications

References 31 publications

Folded Monomers and Hexamers of the Ectodomain of the HIV gp41 Membrane Fusion Protein: Potential Roles in Fusion and Synergy Between the Fusion Peptide, Hairpin, and Membrane-Proximal External Region

Folded Monomers and Hexamers of the Ectodomain of the HIV gp41 Membrane Fusion Protein: Potential Roles in Fusion and Synergy Between the Fusion Peptide, Hairpin, and Membrane-Proximal External Region

Dissociation of the trimeric gp41 ectodomain at the lipid–water interface suggests an active role in HIV-1 Env-mediated membrane fusion

pH-dependent vesicle fusion induced by the ectodomain of the human immunodeficiency virus membrane fusion protein gp41: Two kinetically distinct processes and fully-membrane-associated gp41 with predominant β sheet fusion peptide conformation

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