Designing a Soluble Near Full-length HIV-1 gp41 Trimer

Gao, Guofen; Wieczorek, Lindsay; Peachman, Kristina K.; Polonis, Victoria R.; Alving, Carl R.; Rao, Mangala; Rao, Venigalla B.

doi:10.1074/jbc.m112.424432

Cited by 20 publications

(23 citation statements)

References 73 publications

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“…S2). Higher-order complexes have been reported recently for several ectodomain constructs in the absence of detergent (Gao et al 2013; Banerjee and Weliky 2014) and may reflect non-specific inter-molecular interactions between the membrane proximal regions. In the presence of DPC, the sedimentation velocity experiments for all three ectodomain constructs show the presence of a single species, corresponding to a protein monomer bound to a DPC micelle (Fig.…”

Section: Resultsmentioning

confidence: 64%

Complete dissociation of the HIV-1 gp41 ectodomain and membrane proximal regions upon phospholipid binding

et al. 2015

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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. Strong lipid affinity of the ectodomain suggests that its heptad repeat regions play an active role in destabilizing membranes by directly binding to the lipid bilayers and thereby lowering the free-energy barrier for membrane fusion. In such a model, immediately following the shedding of gp120, the N-heptad and C-heptad helices dissociate and melt into the host cell and viral membranes, respectively, pulling the destabilized membranes into juxtaposition, ready for fusion. Post-fusion, reaching the final 6-helix bundle (6HB) conformation then involves competition between intermolecular interactions needed for formation of the symmetric 6HB trimer and the membrane affinity of gp41's ectodomain, including its membrane-proximal regions. Our solution NMR study of the structural and dynamic properties of three constructs containing the ectodomain of gp41 with and without its membrane-proximal regions suggests that these segments do not form inter-helical interactions until the very late steps of the fusion process. Interactions between the polar termini of the heptad regions, which are not associating with the lipid surface, therefore may constitute the main driving force initiating formation of the final post-fusion states. The absence of significant intermolecular ectodomain interactions in the presence of dodecyl phosphocholine highlights the importance of trimerization of gp41’s transmembrane helix to prevent complete dissociation of the trimer during the course of fusion.

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Section: Resultsmentioning

confidence: 64%

Complete dissociation of the HIV-1 gp41 ectodomain and membrane proximal regions upon phospholipid binding

et al. 2015

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“…If the highly helical Hairpin region is a molecular trimer, the antiparallel FP β sheet could reflect interleaved FPs from two different trimers. This result correlates with predominant hexamers for gp41 ectodomain constructs under some conditions [7,8]. Because of protein aggregation in neutral pH aqueous solution, the SSNMR samples could also have contained a fraction of aggregated protein that was not membrane-associated.…”

Section: Introductionmentioning

confidence: 79%

“…The slow process is attributed to membrane-bound protein oligomers which are the likely equilibrium structural state and which may form through protein diffusion on the membrane surface on the ~200 s time scale of the slow process. For some conditions, the displayed hexamer ≡ two trimer state is the predominant oligomerization state for large ectodomain constructs such as HP [7,8]. …”

Section: Discussionmentioning

confidence: 99%

“…At lower protein concentrations, the ectodomain remains highly helical but is monomeric [6,7]. 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%

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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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“…Further conformational changes occur after co-receptor interaction leading to the opening up of the two long helices HR1 and HR2 of gp41 and insertion of the N-terminal fusion peptide into the host cell membrane [139, 140]. A gp41 prehairpin intermediate [141], a three-stranded coiled coil stabilized by inter-molecular interactions between HR1 helices, is then formed, followed by the disassociation of gp120 subunits from gp41, allowing the HR2 helices to fold back and interact with the HR1 helices. The trimeric HR1 core, the interacting HR2 helices and the intervening loops in between form a hairpin structure of gp41 that is referred to as the six-helix bundle (6HB).…”

Section: Figurementioning

confidence: 99%

HIV-1 Variable Loop 2 and its Importance in HIV-1 Infection and Vaccine Development

Rao¹,

Peachman²,

Kim³

et al. 2013

CHR

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A vaccine that can prevent the transmission of HIV-1 at the site of exposure to the host is one of the best hopes to control the HIV-1 pandemic. The trimeric envelope spike consisting of heterodimers, gp120 and gp41, is essential for virus entry and thus has been a key target for HIV-1 vaccine development. However, it has been extremely difficult to identify the types of antibodies required to block the transmission of various HIV-1 strains and the immunogens that can elicit such antibodies due to the high genetic diversity of the HIV-1 envelope. The modest efficacy of the gp120 HIV-1 vaccine used in the RV144 Thai trial, including the studies on the immune correlates of protection, and the discovery of vaccine-induced immune responses to certain signature regions of the envelope have shown that the gp120 variable loop 2 (V2) is an important region. Since there is evidence that the V2 region interacts with the integrin α4β7 receptor of the host cell, and that this interaction might be important for virus capture, induction of antibodies against V2 loop could be postulated as one of the mechanisms to prevent the acquisition of HIV-1. Immunogens that can induce these antibodies should therefore be taken into consideration when designing HIV-1 vaccine formulations.

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Designing a Soluble Near Full-length HIV-1 gp41 Trimer

Cited by 20 publications

References 73 publications

Complete dissociation of the HIV-1 gp41 ectodomain and membrane proximal regions upon phospholipid binding

Complete dissociation of the HIV-1 gp41 ectodomain and membrane proximal regions upon phospholipid binding

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

HIV-1 Variable Loop 2 and its Importance in HIV-1 Infection and Vaccine Development

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