Nuclear magnetic resonance evidence for retention of a lamellar membrane phase with curvature in the presence of large quantities of the HIV fusion peptide

Gabrys, Charles M.; Yang, Rong; Wasniewski, Christopher M.; Yang, Jun; Canlas, Christian G.; Wang, Qiang; Sun, Yan; Weliky, David P.

doi:10.1016/j.bbamem.2009.07.007

Cited by 20 publications

(45 citation statements)

References 50 publications

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“…The fusion transition state likely contains regions of high membrane curvature and experimental data from SSNMR and other methods support a reduced bending modulus for the membrane in the presence of HFP. 33,34 The equilibrium membrane bilayer structure remains intact with HFP:lipid mole ratio = 0.1 which is consistent with expected evolutionary pressure for a viable host cell. 33 …”

Section: Introductionsupporting

confidence: 75%

Solid-State Nuclear Magnetic Resonance Measurements of HIV Fusion Peptide ¹³CO to Lipid ³¹P Proximities Support Similar Partially Inserted Membrane Locations of the α Helical and β Sheet Peptide Structures

et al. 2013

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Fusion of the HIV membrane and the host cell membrane is an initial step of infection of the host cell. Fusion is catalyzed by gp41 which is an integral membrane protein of HIV. The fusion peptide (FP) is the ~25 N-terminal residues of gp41 and is a domain of gp41 that plays a key role in fusion catalysis likely through interaction with the host cell membrane. Much of our understanding of the FP domain has been accomplished with studies of “HFP”, ie. a ~25-residue peptide composed of the FP sequence but lacking the rest of gp41. HFP catalyzes fusion between membrane vesicles and serves as a model system to understand fusion catalysis. HFP binds to membranes and the membrane location of HFP is likely a significant determinant of fusion catalysis perhaps because the consequent membrane perturbation reduces the fusion activation energy. In the present study, many HFPs were synthesized and differed in the residue position that was 13CO backbone labeled. Samples were then prepared that each contained a singly 13CO labeled HFP incorporated into membranes which lacked cholesterol. HFP had distinct molecular populations with either α helical or oligomeric β sheet structure. Proximity between the HFP 13CO nuclei and 31P nuclei in the membrane headgroups was probed by solid-state NMR (SSNMR) rotational-echo double-resonance (REDOR) measurements. For many samples, there were distinct 13CO shifts for the α helical and β sheet structures so that the proximities to 31P nuclei could be determined for each structure. Data from several differently labeled HFPs were then incorporated into a membrane location model for the particular structure. In addition to the 13CO labeled residue position, the HFPs also differed in sequence and/or chemical structure. “HFPmn” was a linear peptide that contained the 23 N-terminal residues of gp41. “HFPmn_V2E” contained the V2E mutation which for HIV leads to greatly reduced extent of fusion and infection. The present study shows that HFPmn_V2E induces much less vesicle fusion than HFPmn. “HFPtr” contained three strands with HFPmn sequence that were chemically cross-linked near their C-termini. HFPtr mimics the trimeric topology of gp41 and induces much more rapid and extensive vesicle fusion than HFPmn. For HFPmn and HFPtr, well-resolved α and β peaks were observed for A6-, L9-, and L12-labeled samples. For each of these samples, there were similar HFP 13CO to lipid 31P proximities in the α and β structures which evidenced comparable membrane locations of the HFP in either structure including insertion into a single membrane leaflet. The data were also consistent with deeper insertion of HFPtr relative to HFPmn in both the α and β structures. The results supported a strong correlation between the membrane insertion depth of the HFP and its fusogenicity. More generally, the results supported membrane location of the HFP as an important determinant of its fusogenicity. The deep insertion of HFPtr in both the α and β structures provides the most relevant membrane location of the FP for HIV gp41-cat...

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Section: Introductionsupporting

confidence: 75%

Solid-State Nuclear Magnetic Resonance Measurements of HIV Fusion Peptide ¹³CO to Lipid ³¹P Proximities Support Similar Partially Inserted Membrane Locations of the α Helical and β Sheet Peptide Structures

et al. 2013

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“…The oligomer with β FP’s is proposed as the catalytic structure of the slow fusion process. The FP may locally perturb the surrounding bilayer structure of the lipids (at equilibrium) so that there is smaller activation energy to the fusion transition state in which lipid structures are also disordered relative to an unperturbed bilayer [34–37]. …”

Section: Discussionmentioning

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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“…13 CO signal. Bilayer phase is retained for ether-linked lipids with cholesterol and with HFP (34–36). In addition, membrane-associated HFP has predominant β sheet structure in either ester-linked lipid + cholesterol or ether-linked lipid + cholesterol compositions (30).…”

Section: Methodsmentioning

confidence: 99%

Major Antiparallel and Minor Parallel β Sheet Populations Detected in the Membrane-Associated Human Immunodeficiency Virus Fusion Peptide

Schmick

Weliky

2010

Biochemistry

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The HIV gp41 protein catalyzes fusion between viral and host cell membranes and its apolar N-terminal region or “fusion peptide” binds to the host cell membrane and plays a key role in fusion. “HFP” is a construct containing the fusion peptide sequence, induces membrane vesicle fusion, and is an important fusion model system. Earlier solid-state NMR (SSNMR) studies showed that when HFP is associated with membranes with ~30 mol% cholesterol, the first sixteen residues have predominant β strand secondary structure and a fraction of the strands form antiparallel β sheet structure with residue 16→1/1→16 or 17→1/1→17 registries of adjacent strands. In some contrast, other SSNMR and infrared studies have been interpreted to support a large fraction of approximately in-register parallel registry of adjacent strands. However, the samples had extensive isotopic labeling and other structural models were also consistent with the data. The present SSNMR study uses sparse labeling schemes that reduce ambiguity in the determination of the fraction of HFP molecules with parallel β registry. Quantitative analysis of the data shows that the parallel fraction is at most 0.15 with a much greater fraction of antiparallel 16→1/1→16 and 17→1/1→17 registries. These data strongly support a model of HFP-induced vesicle fusion caused by antiparallel rather than parallel registries and provide insight into the arrangement of gp41 molecules during HIV/host cell fusion. This study is an example of quantitative determination of a complex structural distribution by SSNMR including experimentally-validated inclusion of natural abundance contributions to the SSNMR data.

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Nuclear magnetic resonance evidence for retention of a lamellar membrane phase with curvature in the presence of large quantities of the HIV fusion peptide

Cited by 20 publications

References 50 publications

Solid-State Nuclear Magnetic Resonance Measurements of HIV Fusion Peptide ¹³CO to Lipid ³¹P Proximities Support Similar Partially Inserted Membrane Locations of the α Helical and β Sheet Peptide Structures

Solid-State Nuclear Magnetic Resonance Measurements of HIV Fusion Peptide ¹³CO to Lipid ³¹P Proximities Support Similar Partially Inserted Membrane Locations of the α Helical and β Sheet Peptide Structures

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

Major Antiparallel and Minor Parallel β Sheet Populations Detected in the Membrane-Associated Human Immunodeficiency Virus Fusion Peptide

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Nuclear magnetic resonance evidence for retention of a lamellar membrane phase with curvature in the presence of large quantities of the HIV fusion peptide

Cited by 20 publications

References 50 publications

Solid-State Nuclear Magnetic Resonance Measurements of HIV Fusion Peptide 13CO to Lipid 31P Proximities Support Similar Partially Inserted Membrane Locations of the α Helical and β Sheet Peptide Structures

Solid-State Nuclear Magnetic Resonance Measurements of HIV Fusion Peptide 13CO to Lipid 31P Proximities Support Similar Partially Inserted Membrane Locations of the α Helical and β Sheet Peptide Structures

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

Major Antiparallel and Minor Parallel β Sheet Populations Detected in the Membrane-Associated Human Immunodeficiency Virus Fusion Peptide

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Solid-State Nuclear Magnetic Resonance Measurements of HIV Fusion Peptide ¹³CO to Lipid ³¹P Proximities Support Similar Partially Inserted Membrane Locations of the α Helical and β Sheet Peptide Structures

Solid-State Nuclear Magnetic Resonance Measurements of HIV Fusion Peptide ¹³CO to Lipid ³¹P Proximities Support Similar Partially Inserted Membrane Locations of the α Helical and β Sheet Peptide Structures