Solid-State NMR Spectroscopy of the HIV gp41 Membrane Fusion Protein Supports Intermolecular Antiparallel β Sheet Fusion Peptide Structure in the Final Six-Helix Bundle State

Sackett, Kelly; Nethercott, Matthew J.; Zheng, Zhaoxiong; Weliky, David P.

doi:10.1016/j.jmb.2013.11.010

Cited by 35 publications

(43 citation statements)

References 55 publications

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“…26 A membrane-associated gp41 hexamer is consistent with the antiparallel β sheet FP structure in the membrane-associated gp41 ectodomain observed by SSNMR. 24 Such structure is consistent with interleaved FP strands from two trimers (Figure 11C).…”

Section: Discussionsupporting

confidence: 75%

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: 75%

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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“…At either low or neutral pH, the major population of membrane-associated FP-HP molecules have β FP structure. Previous SSNMR showed this was an intermolecular antiparallel β sheet, likely an equilibrium structure as the SSNMR is done after ~1 day of sample preparation [20,33]. The oligomer with β FP’s is proposed as the catalytic structure of the slow fusion process.…”

Section: Discussionmentioning

confidence: 99%

“…HP and FP-HP proteins were produced and purified as previously described [13,15,20]. Briefly, FP was synthesized by t -boc chemical synthesis and the subsequent cleavage with hydrogen fluoride was done by Midwest Biotech.…”

Section: Methodsmentioning

confidence: 99%

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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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“…This is very much the case for membrane proteins [14-17], as the stability and folding of a membrane protein can be controlled by intermolecular interactions. For example, most membrane proteins are unstable in solution but they fold natively in a lipid bilayer environment to form stable structures.…”

Section: Introductionmentioning

confidence: 99%

Cellular solid-state NMR investigation of a membrane protein using dynamic nuclear polarization

Yamamoto

Caporini

et al. 2015

Biochimica et Biophysica Acta (BBA) - Biomembranes

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While an increasing number of structural biology studies successfully demonstrate the power of high-resolution structures and dynamics of membrane proteins in fully understanding their function, there is considerable interest in developing NMR approaches to obtain such information in a cellular setting. As long as the proteins inside the living cell tumble rapidly in the NMR timescale, recently developed in-cell solution NMR approaches can be applied towards the determination of 3D structural information. However, there are numerous challenges that need to be overcome to study membrane proteins inside a cell. Research in our laboratory is focused on developing a combination of solid-state NMR and biological approaches to overcome these challenges with a specific emphasis on obtaining high-resolution structural insights into electron transfer biological processes mediated by membrane-bound proteins like mammalian cytochrome b5, cytochrome P450 and cytochrome P450 reductase. In this study, we demonstrate the feasibility of using the signal-enhancement rendered by dynamic nuclear polarization (DNP) magic angle spinning (MAS) NMR spectroscopy for in-cell studies on a membrane-anchored protein. Our experimental results obtained from 13C-labeled membrane-anchored cytochrome b5 in native Escherichia coli cells show a ~16-fold DNP signal enhancement (ε). Further, results obtained from a 2D 13C/13C chemical shift correlation MAS experiment demonstrates that it is highly possible to suppress the background signals from other cellular contents for high-resolution structural studies on membrane proteins. We believe that this study would pave new avenues for high-resolution 3D structural studies on a variety of membrane-associated proteins and their complexes in the cellular context to fully understand their functional roles in physiological processes.

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Solid-State NMR Spectroscopy of the HIV gp41 Membrane Fusion Protein Supports Intermolecular Antiparallel β Sheet Fusion Peptide Structure in the Final Six-Helix Bundle State

Cited by 35 publications

References 55 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

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

Cellular solid-state NMR investigation of a membrane protein using dynamic nuclear polarization

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