Membrane proteins are of outstanding importance in biology, drug discovery and vaccination. A common limiting factor in research and applications involving membrane proteins is the ability to solubilize and stabilize membrane proteins. Although detergents represent the major means for solubilizing membrane proteins, they are often associated with protein instability and poor applicability in structural and biophysical studies. Here, we present a novel lipoprotein nanoparticle system that allows for the reconstitution of membrane proteins into a lipid environment that is stabilized by a scaffold of Saposin proteins. We showcase the applicability of the method on two purified membrane protein complexes as well as the direct solubilization and nanoparticle-incorporation of a viral membrane protein complex from the virus membrane. We also demonstrate that this lipid nanoparticle methodology facilitates high-resolution structural studies of membrane proteins in a lipid environment by single-particle electron cryo-microscopy (cryo-EM) and allows for the stabilization of the HIV-envelope glycoprotein in a functional state.

The HIV-1 spike is a trimer of the transmembrane gp41 and the peripheral gp120 subunit pair. It is activated for virus-cell membrane fusion by binding sequentially to CD4 and to a chemokine receptor. Here we have studied the structural transition of the trimeric spike during the activation process. We solubilized and isolated unliganded and CD4-bound spikes from virus-like particles and used cryoelectron microscopy to reconstruct their 3D structures. In order to increase the yield and stability of the spike, we used an endodomain deleted and gp120-gp41 disulfide-linked variant. The unliganded spike displayed a hollow cage-like structure where the gp120-gp41 protomeric units formed a roof and bottom, and separated lobes and legs on the sides. The tripod structure was verified by fitting the recent atomic core structure of gp120 with intact N-and C-terminal ends into the spike density map. This defined the lobe as gp120 core, showed that the legs contained the polypeptide termini, and suggested the deleted variable loops V1/V2 and V3 to occupy the roof and gp41 the bottom. CD4 binding shifted the roof density peripherally and condensed the bottom density centrally. Fitting with a V3 containing gp120 core suggested that the V1/V2 loops in the roof were displaced laterally and the V3 lifted up, while the core and leg were kept in place. The loop displacements probably prepared the spike for coreceptor interaction and roof opening so that a new fusion-active gp41 structure, assembled at the center of the cage bottom, could reach the target membrane.retrovirus spike | receptor triggering | cryo-EM | single particle imaging | EMAN T he HIV-1 spike facilitates entry of the virus into the cell by mediating fusion between the viral and the cell membranes. It also represents the target for neutralizing antibodies of the host. The spike is assembled from three copies of a transmembrane precursor glycoprotein, gp160, in the endoplasmic reticulum of the infected cell and is activated by a series of structural transitions (1-3). When the spike passes trans Golgi, on its way to the cell surface, gp160 is cleaved by furin into gp41 and gp120, which remain noncovalently linked (4). The cleavage positions the fusion peptide at the N terminus of gp41 and primes the spike for fusion activation. In the virus the gp120 subunits suppress the fusion activity of the gp41 subunits until structurally changed by receptor interactions, first with CD4 and then with the chemokine coreceptor (5-9). The gp41 subunits induce membrane fusion through refolding into a more stabile form. According to the prevailing model, the gp41 first targets the cell membrane with its fusion peptide and then folds back on itself dragging the virus and the cell membranes together for fusion (10). Characteristic for the gp41 ectodomain is two α-helical regions (N and C helices) separated by a small disulfide loop, CX 5 C. Peptides corresponding to the helical regions form a stable complex in solution and the crystal structure shows a bundle of six helices, where thr...

The Env protein of murine leukemia virus matures by two cleavage events. First, cellular furin separates the receptor binding surface (SU) subunit from the fusion-active transmembrane (TM) subunit and then, in the newly assembled particle, the viral protease removes a 16-residue peptide, the R-peptide from the endodomain of the TM. Both cleavage events are required to prime the Env for receptor-triggered activation. Cryoelectron microscopy (cryo-EM) analyses have shown that the mature Env forms an open cage-like structure composed of three SU-TM complexes, where the TM subunits formed separated Env legs. Here we have studied the structure of the R-peptide precursor Env by cryo-EM. TM cleavage in Moloney murine leukemia virus was inhibited by amprenavir, and the Envs were solubilized in Triton X-100 and isolated by sedimentation in a sucrose gradient. We found that the legs of the R-peptide Env were held together by trimeric interactions at the very bottom of the Env. This suggested that the R-peptide ties the TM legs together and that this prevents the activation of the TM for fusion. The model was supported by further cryo-EM studies using an R-peptide Env mutant that was fusion-competent despite an uncleaved R-peptide. The Env legs of this mutant were found to be separated, like in the mature Env. This shows that it is the TM leg separation, normally caused by R-peptide cleavage, that primes the Env for receptor triggering.three-dimensional structure | retrovirus | spike protein T he spike protein Env on the surface of the retrovirus murine leukemia virus (MLV) matures by two proteolytic cleavage events mediated by cellular furin and the viral protease (1-3). Env is composed of an 80-kDa transmembrane precursor protein in the rough endoplasmic reticulum of the infected cell (4, 5). Here it trimerizes before it is routed to the cell surface for assembly with the internal Gag and GagPol precursors into virus particles in a budding process (6). The furin cleavage of Env occurs in the trans-Golgi, and it separates the surface (SU) subunit from the transmembrane (TM) (Pr15E) subunit. This cleavage also releases the viral fusion peptide at the N-terminal end of the TM. The viral protease cleavage occurs after virus assembly in the newly formed particle. It removes a 16-residuelong peptide, the R-peptide from the C terminus of the TM, forming p15E (7). Not until this second cleavage is completed is Env primed for receptor-mediated triggering into further conformations that can direct virus entry through fusion of the viral membrane with the cell membrane (8, 9). The viral protease also cleaves the Gag and GagPol precursors into their mature proteins and enzymes.The structure of the MLV Env has been studied by cryoelectron microscopy (cryo-EM) both in intact particles and as solubilized trimers (10, 11). It reveals a remarkably open structure with separated legs. The protomer of the solubilized Env is formed from three protrusions-upper, middle, and lower. Together, they encage a large central cavity, with the top pro...

Fusion of the membrane of the Moloney murine leukemia virus (Mo-MLV) Env protein is facilitated by cleavage of the R peptide from the cytoplasmic tail of its TM subunit, but the mechanism for this effect has remained obscure. The fusion is also controlled by the isomerization of the intersubunit disulfide of the Env SU-TM complex. In the present study, we used several R-peptide-cleavage-inhibited virus mutants to show that the R peptide suppresses the isomerization reaction in both in vitro and in vivo assays. Thus, the R peptide affects early steps in the activation pathway of murine leukemia virus Env.During maturation of the Moloney murine leukemia virus (Mo-MLV), the viral protease cleaves a 16-residue-long peptide, the R peptide, from the cytoplasmic tail of the TM subunit of the membrane fusion protein Env (5, 6, 17). The cleavage potentiates the receptor-induced fusion activation in Env, but the mechanism for this effect has remained obscure (7,8,15,16,18). Similar regulation has been observed in other gammaretroviruses and in the Mason-Pfizer monkey betaretrovirus (2, 3). Recently, it was shown that the activation of MLV fusion is also controlled by receptor-induced isomerization of the intersubunit disulfide of the Env SU-TM subunit complex (14,19). Here, we studied whether R-peptide cleavage facilitates the isomerization.R-peptide cleavage site mutants, shown to inhibit cleavage in earlier studies, were created at the P1 (L649V, L649R, and L649I) and P1Ј (V650I) positions by PCR mutagenesis, using Mo-MLV proviral DNA (4,8,16,18). Corresponding particles were produced and labeled with 50 Ci/ml [ 35 S]Cys in calcium phosphate precipitate-transfected HEK 293T cells. Analysis by reducing sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) of sedimentation-purified viruses that had been lysed in buffer containing 50 mM Tris-HCl (pH 7.5), 150 mM NaCl, and 2 mM EDTA and immunoprecipitated with anti-MLV polyclonal antibody (pAb) (HE863; Viromed Biosafety Laboratories) showed that the mutant Env's, with the exception of V650I, incorporated with wild-type (wt) efficiency and were significantly inhibited in R-peptide cleavage, i.e., in cleavage of the Pr15E form of TM into the p15E form (Table 1). Incorporation of Env-V650I was reduced to 63%, and the R peptide was cleaved as in the wt. The infectivities of the mutants were analyzed in XC cells by using MLV p12 monoclonal antibody (Ab) 548 (B. W. Chesebro, Rocky Mountain Laboratories). Cell binding was augmented by centrifugation of the virus-cell sample at 850 ϫ g for 1 h at 4°C in the presence of 8 g/ml polybrene. The infectivities of the mutants were found to correlate with the biochemical findings (Table 1). This was also the case with their fusion efficiencies, which were tested in an XC cell-to-cell fusion-from-without assay (19). Thus, all mutants but V650I were inhibited in R-peptide cleavage and membrane fusion. Notably, the cleavage inhibition was only partial, and the L649V mutant contained a significant fraction (about 30%) of R-pept...

The surface (SU) and transmembrane (TM) subunits of Moloney murine leukemia virus (Mo-MLV) Env are disulfide linked. The linking cysteine in SU is part of a conserved CXXC motif in which the other cysteine carries a free thiol. Recently, we showed that receptor binding activates its free thiol to isomerize the intersubunit disulfide bond into a disulfide within the motif instead (M. Wallin, M. Ekström and H. Garoff, EMBO J. 23: [54][55][56][57][58][59][60][61][62][63][64][65] 2004). This facilitated SU dissociation and activation of TM for membrane fusion. The evidence was mainly based on the finding that alkylation of the CXXC-thiol prevented isomerization. This arrested membrane fusion, but the activity could be rescued by cleaving the intersubunit disulfide bond with dithiothreitol (DTT). Here, we demonstrate directly that receptor binding causes SU-TM disulfide bond isomerization in a subfraction of the viral Envs. The kinetics of the isomerization followed that of virus-cell membrane fusion. Arresting the fusion with lysophosphatidylcholine did not arrest isomerization, suggesting that isomerization precedes the hemifusion stage of fusion. Our earlier finding that native Env was not possible to alkylate but required isomerization induction by receptor binding intimated that alkylation trapped an intermediate form of Env. To further clarify this possibility, we analyzed the kinetics by which the alkylationsensitive Env was generated during fusion. We found that it followed the fusion kinetics. In contrast, the release of fusion from alkylated, isomerization-blocked virus by DTT reduction of the SU-TM disulfide bond was much faster. These results suggest that the alkylation-sensitive form of Env is a true intermediate in the fusion activation pathway of Env.The retroviruses enter cells by fusing their membranes with that of the target cell. The fusion is facilitated by the activity of the viral glycoprotein (14). The latter is composed of three copies of a two-subunit protein, Env. One of the subunits, surface (SU), has a peripheral topology, and the other, transmembrane (TM), has a transmembrane topology. The membrane fusion activity is loaded into TM but suppressed by the associated SU. The fusion is triggered when the virus binds to its cell surface receptor via SU. An exception is avian sarcoma and leukosis virus, in which complete triggering demands both receptor binding and low pH (31). The fusion activation is assumed to follow the mechanism revealed in influenza hemagglutinin (HA) (39). According to this model, the TM subunit of the retrovirus persists in a metastable state in the native Env and refolds upon receptor-induced displacement of SU. The refolding of TM involves the exposure of its N-terminal fusion peptide at the membrane-distal part of the molecule, where it can interact with the cell membrane. Further, the TM undergoes a jackknife-like backfolding. This brings the C-terminal transmembrane peptide of TM with the attached viral membrane toward the N-terminal fusion peptide in the cell memb...

cThe HIV-1 spike is composed of three protomeric units, each containing a peripheral gp120 and a transmembrane gp41 subunit. Binding to the CD4 and the chemokine receptors triggers them to mediate virus entry into cells by membrane fusion. The spikes also represent the major target for neutralizing antibodies (Abs) against the virus. We have studied how two related broadly neutralizing Abs, PG9 and PG16, react with the spike. Unexpectedly, this also suggested how the functions of the individual protomers in the spike depend on each other. The Abs have been shown to bind the V1/V2 loops of gp120, located at the top of the spike. T he HIV-1 spike is a trimer where the protomeric unit is composed of the noncovalently linked peripheral (gp120) and transmembrane (gp41) subunits (1-3). The spike mediates entry of the virus into the cell through membrane fusion. In this process, gp120 binds successively to the primary, CD4, and the secondary, chemokine receptors on the cell surface. This activates gp41 to interact with the lipid bilayer of the cell via its fusion peptide (4, 5). The back-folding of the gp41 subunits from a trimeric prehairpin to a hairpin structure then approaches the viral and the cell membranes so they can merge and allow the viral capsid with the viral genome to enter into the cell (6-8).It has been shown that binding of CD4 induces changes in the gp120 structure that result in formation of the binding site for the secondary receptor and that changes induced by both receptors release the gp120 constraints on gp41 activation (1, 3, 9-11). However, an important question concerns the possible coordination of the activating processes in the individual protomers of the trimeric spike. Are the activating changes in the protomers linked to each other and therefore have to occur simultaneously? A coordinated process would ensure the formation of symmetrical intermediate forms of the trimeric spike, which should be important for their stability and function.The spike is also the target for neutralizing antibodies (NAbs). Several broadly neutralizing antibodies (bNAbs) against HIV-1 have been characterized (12)(13)(14)(15)(16)(17)(18)(19). The elucidation of their neutralization mechanisms should be useful for the development of an HIV-1 vaccine and also to explain the spike activation mechanism. Some bNAbs like 2G12 and VRC01stabilize the native unliganded conformation of the spike and thereby inhibit spike activation (10,20). Most interestingly, single Ab binding seems to be sufficient for neutralization (21). This can be explained assuming a coordinated activation mechanism of the three protomers of the spike.In this case, binding of a single stabilizing bNAb to one protomer will also prevent the activation of the unliganded protomers.In the present study we have studied the neutralization mechanism of the bNAbs PG9 and PG16 (17,22). These are somatically related Abs that neutralize the majority of the HIV-1 strains. They are sensitive to changes in the gp120 variable (V) loops V1/V2 and also V3, require t...

Integral membrane proteins (IMPs) are central to many physiological processes and represent ∼60% of current drug targets. An intricate interplay with the lipid molecules in the cell membrane is known to influence the stability, structure and function of IMPs. Detergents are commonly used to solubilize and extract IMPs from cell membranes. However, due to the loss of the lipid environment, IMPs usually tend to be unstable and lose function in the continuous presence of detergent. To overcome this problem, various technologies have been developed, including protein engineering by mutagenesis to improve IMP stability, as well as methods to reconstitute IMPs into detergent-free entities, such as nanodiscs based on apolipoprotein A or its membrane scaffold protein (MSP) derivatives, amphipols, and styrene-maleic acid copolymer-lipid particles (SMALPs). Although significant progress has been made in this field, working with inherently unstable human IMP targets (e.g., GPCRs, ion channels and transporters) remains a challenging task. Here, we present a novel methodology, termed DirectMX (for direct membrane extraction), taking advantage of the saposin-lipoprotein (Salipro) nanoparticle technology to reconstitute fragile IMPs directly from human crude cell membranes. We demonstrate the applicability of the DirectMX methodology by the reconstitution of a human solute carrier transporter and a wild-type GPCR belonging to the human chemokine receptor (CKR) family. We envision that DirectMX bears the potential to enable studies of IMPs that so far remained inaccessible to other solubilization, stabilization or reconstitution methods.

The trimeric Moloney murine leukemia virus Env protein matures by two proteolytic cleavages. First, furin cleaves the Env precursor into the surface (SU) and transmembrane (TM) subunits in the cell and then the viral protease cleaves the R-peptide from TM in new virus. Here we analyzed the structure of the furin precursor, by cryoelectron microscopy. We transfected 293T cells with a furin cleavage site provirus mutant, R466G/K468G, and produced the virus in the presence of amprenavir to also inhibit the R-peptide cleavage. Although Env incorporation into particles was inhibited, enough precursor could be isolated and analyzed by cryoelectron microscopy to yield a 3D structure at 22 Å resolution. This showed an open cage-like structure like that of the R-peptide precursor and the mature Env described before. However, the middle protrusion of the protomeric unit, so prominently pointing out from the side of the more mature forms of the Env, was absent. Instead, there was extra density in the top protrusion. This suggested that the C-terminal SU domain was associated alongside the receptor binding N-terminal SU domain in the furin precursor. This was supported by mapping with a SU C-terminal domain-specific antigen binding fragment. We concluded that furin cleavage not only separates the subunits and liberates the fusion peptide at the end of TM but also allows the C-terminal domain to relocate into a peripheral position. This conformational change might explain how the C-terminal domain of SU gains the potential to undergo disulfide isomerization, an event that facilitates membrane fusion.