Interplay Between Membrane Curvature and Protein Conformational Equilibrium Investigated by Solid-State NMR

Interplay between membrane curvature and protein conformational equilibrium investigated by solid-state NMR

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Citation Liao, Shu Y., Myungwoon Lee, and Mei Hong, "Interplay between membrane curvature and protein conformational equilibrium investigated by solid-state NMR." Journal of structural biology 206, 1 (April 2019): p. 20-28 doi 10.1016/J.JSB.2018.02.007 ©2019 Author(s)

As Published 10.1016/J.JSB.2018.02.007

Publisher Elsevier BV

Version Author's final manuscript

Citable link https://hdl.handle.net/1721.1/124604

Detailed Terms http://creativecommons.org/licenses/by-nc-nd/4.0/ HHS Public Access Author manuscript

Author ManuscriptAuthor Manuscript Author J Struct Manuscript Author Biol. Author manuscript; Manuscript Author available in PMC 2020 April 01. Published in final edited form as: J Struct Biol. 2019 April 01; 206(1): 20–28. doi:10.1016/j.jsb.2018.02.007.

Interplay Between Membrane Curvature and Protein Conformational Equilibrium Investigated by Solid-State NMR

Shu Y. Liao, Myungwoon Lee, and Mei Hong* Department of Chemistry, Massachusetts Institute of Technology, Cambridge, MA 02139

Abstract Many membrane proteins sense and induce membrane curvature for function, but structural information about how proteins modulate their structures to cause membrane curvature is sparse. We review our recent solid-state NMR studies of two virus membrane proteins whose conformational equilibrium is tightly coupled to membrane curvature. The influenza M2 proton channel has a drug-binding site in the transmembrane (TM) pore. Previous chemical shift data indicated that this pore-binding site is lost in an M2 construct that contains the TM domain and a curvature-inducing amphipathic helix. We have now obtained chemical shift perturbation, protein- drug proximity, and drug orientation data that indicate that the pore-binding site is restored when the full cytoplasmic domain is present. This finding indicates that the curvature-inducing amphipathic helix distorts the TM structure to interfere with drug binding, while the cytoplasmic tail attenuates this effect. In the second example, we review our studies of a parainfluenza virus fusion protein that merges the cell membrane and the virus envelope during virus entry. Chemical shifts of two hydrophobic domains of the protein indicate that both domains have membrane- dependent backbone conformations, with the β-strand structure dominating in negative-curvature phosphatidylethanolamine (PE) membranes. 31P NMR spectra and 1H-31P correlation spectra indicate that the β-strand-rich conformation induces saddle-splay curvature to PE membranes and dehydrates them, thus stabilizing the hemifusion state. These results highlight the indispensable role of solid-state NMR to simultaneously determine membrane protein structures and characterize the membrane curvature in which these protein structures exist.

Introduction Membrane curvature is generated during many biological processes such as virus-cell fusion for virus entry, membrane scission for virus budding, and intracellular vesicle trafficking between organelles (McMahon and Gallop, 2005). Membrane proteins can both sense and cause membrane curvature. However, the interplay between protein structure and membrane- curvature sensing and generation are poorly understood due to the difficulty of studying protein structures at atomic resolution in native phospholipid membranes. Here we review our recent studies and present new data of two membrane proteins that modulate their

*Corresponding Author: Mei Hong, Tel: 617-253-5521, [email protected]. Publisher's Disclaimer: This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. Liao et al. Page 2

structures to sense and cause membrane curvature. The first example is the influenza M2 Author ManuscriptAuthor Manuscript Author Manuscript Author Manuscript Author protein, which is well known to be a drug-targeted proton channel (Cady et al., 2009b; Hong and DeGrado, 2012) but also has a second function of causing membrane scission during influenza virus budding (Rossman and Lamb, 2011). The second protein is the parainfluenza virus 5 (PIV5) fusion protein F, a member of the class-I viral fusion proteins (Lamb and Jardetzky, 2007). Viral fusion proteins merge the cell membrane and the virus lipid envelope to enable virus entry into cells (Harrison, 2008). We review our recent solid-state NMR results of the conformations of the N-terminal fusion peptide (FP) and the C-terminal TM domain (TMD) of PIV5 F. Both domains exhibit striking membrane-dependent structures, with the β-sheet structure correlated with membrane-curvature induction and membrane- surface dehydration, which are necessary for virus-cell fusion.

The influenza A M2 protein forms a drug-targeted proton channel in the virus envelope that is important for the virus lifecycle (Cady et al., 2009b; Hong and DeGrado, 2012; Pinto and Lamb, 2006). The proton-channel activity is chiefly carried out by the transmembrane (TM) domain (Ma et al., 2009) while the membrane scission function is carried out by an amphipathic helix (AH) C-terminal to the TM domain (Rossman et al., 2010). Solid-state NMR and X-ray crystallographic data have shown that the amantadine family of antiviral drugs binds the N-terminal pore of the channel near S31 (Andreas et al., 2013; Cady et al., 2011b; Cady et al., 2010; Stouffer et al., 2008). In addition there is a second, low-affinity, binding site on the lipid-facing surface of the C-terminal end of the TM helix (Schnell and Chou, 2008), which was first observed in DHPC-micelle bound M2. The inhibition mechanism of amantadine involves dehydration of the pore (Luo and Hong, 2010), which prevents His37 protonation, in turn blocking channel opening (Hu et al., 2012; Williams et al., 2013). However, there remains an unsolved puzzle that drug binding appears to depend on the protein construct length (Cady et al., 2011a). While the pore binding site was first determined in the TM peptide (residues 22-46) reconstituted in phosphocholine bilayers such as DMPC and DLPC (Cady et al., 2009a; Cady et al., 2010), a longer M2 construct (residues 21-61) that contains both the TM helix and the amphipathic helix (AH) shows attenuated drug binding in DMPC bilayers and loss of drug binding in a virus-mimetic lipid membrane (VM+). These are manifested by the lack of chemical shift perturbations of key TM residues such as S31 and G34. The loss of drug binding to the pore is reminiscent of the absence of drug binding to the pore of micelle-bound TM-AH M2 (Schnell and Chou, 2008), but unlike DHPC micelles, the VM+ membrane contains POPC, POPE, sphingomyelin, and cholesterol, which mimic the composition of virus lipid envelopes (Cady et al., 2011a; Luo et al., 2009). Because electron microscopy data showed that the AH causes significant membrane curvature to mediate membrane scission (Rossman et al., 2010), we hypothesized that the curvature-inducing ability of the AH may have altered the TM four-helix bundle structure in the cholesterol-containing membrane to prevent drug binding. If this hypothesis is correct, then the cytoplasmic tail of M2, which spans residues 62-97 and is dynamically disordered (Kwon et al., 2015; Liao et al., 2013), may counter the curvature-inducing ability of the AH and restore drug binding. This motivated us to study amantadine binding to the full-cytoplasmic-containing M2.

We have now measured 13C and 15N chemical shifts, protein-drug dipolar couplings, and drug orientation in M2(21-97). This construct contains the full cytoplasmic domain and has

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been shown to have the same proton-channel activity as full-length M2 (Ma et al., 2009). We Author ManuscriptAuthor Manuscript Author Manuscript Author Manuscript Author show that indeed amantadine binds the pore of M2(21-97) in both DMPC and VM+ membranes, thus proving that the cytoplasmic tail restores the TM conformation to be competent for drug binding. These results solve the previous puzzle and indicate that the proton-channel function and the membrane scission function are inter-related through drug binding to the TM pore.

Materials and Methods Expression, purification and membrane reconstitution of M2(21-97) M2(21-97) was cloned and expressed in E. Coli as described before (Liao et al., 2015). The construct has two mutations, H57Q and H90Q, to make His37 the only histidine in the protein. The protein was 13C, 15N-labeled at His, Ala, Gly and Ser by adding 100 mg/L of each amino acid to the M9 media. The protein was purified from inclusion bodies using Ni2+ affinity column chromatography, and an N-terminal His-tag was cleaved using TEV protease. The cleaved product was purified by reverse-phase HPLC and the purity was verified by electrospray ionization mass spectrometry. Isotopically labeled M2(21-97) was reconstituted into DMPC and VM+ membranes by octylglucoside dialysis (Liao et al., 2015). The proteoliposomes for SSNMR experiments have a protein: lipid molar ratio of 1: 22.5 and the pellets contain ~40 wt% water. The pH of the samples was pH 7.0 for the DMPC membrane and 8.5 for the VM+ membrane. For samples containing perdeuterated drug, d15-amantadine(Amt) in a water/methanol solution was titrated into the membrane pellet to reach a drug: tetramer ratio of 5: 1, then the membrane pellet was lyophilized and rehydrated with 2H-depleted water. The moderate excess of drug compared to the stoichiometric ratio of 1: 1 ensures saturation of the pore binding site, since amantadine also has affinity for the lipid membrane (Cady et al., 2010). The moderate drug excess does not affect the interpretation of chemical shift perturbations of key pore-lining TM residues, and is quantitatively taken into account in the interpretation of the 2H NMR spectra.

Solid-state NMR experiments Solid-state NMR spectra of M2(21-97) were measured at 400 (9.4 Tesla), 800 (18.8 Tesla) and 900 MHz (21.1 Tesla) NMR spectrometers. All 13C chemical shifts were referenced to 15 the adamantane CH2 chemical shift at 38.48 ppm on the tetramethylsilane scale. N chemical shifts were referenced to the 15N signal of N-acetylvaline at 122.0 ppm on the liquid ammonia scale. 1D 13C cross-polarization (CP) spectra were measured under 14.5– 16.0 kHz MAS from −20°C to 25°C. 2D 15N-13C and 13C-13C correlation spectra were measured on the 800 MHz and 900 MHz spectrometers using 3.2 mm MAS probes. 2D 13C-13C DARR spectra used mixing times of 60 ms and 100 ms, while the 15N -13C correlation spectra used REDOR mixing times of 0.83 – 0.95 ms. 13C-2H REDOR spectra were measured at an MAS frequency of 4250 Hz at −30°C on the 400 MHz spectrometer using a 4 mm MAS probe. The experiment used a Gaussian 13C 180 pulse of 800 μs in the center of the REDOR mixing period to remove the 13C-13C J-coupling and increase the 13 C 2 T2 relaxation times (Jaroniec et al., 2001). Multiple H inversion pulses used the composite 90°180°90° scheme with a 90° pulse length of 6 μs. This composite pulse not only compensates for flip-angle imperfections (Sinha et al., 2004) but also speeds up 13C-2H

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REDOR dephasing and causes it to decay below 0.33 (Sack et al., 1999), thus facilitating Author ManuscriptAuthor Manuscript Author Manuscript Author Manuscript Author dipolar coupling measurement. These two improvements result from reduction of double- quantum quadrupolar coupling terms and introduction of single-quantum terms that involve 2 the ms = 0 state of the H spin angular momentum in the average dipolar Hamiltonian (Sack et al., 1999).

Static 2H quadrupolar spectra of M2(21-97) were measured on the 400 MHz spectrometer at 30°C using a quadrupolar-echo sequence with 50 μs and 36 μs delays before and after the second 90° pulse. The 2H 90° pulse length was 5 μs and the recycle delay was 0.5 s. The numbers of scans ranged from 120,000 to 256,000 to obtain sufficient sensitivity to detect low-intensity quadrupolar coupling components. The time signals were left-shifted manually to capture the echo maximum before Fourier transformation to produce artifact-free 2H spectra. The spectra were simulated in MATLAB using the software EXPRESS (Vold and Hoatson, 2009) to obtain accurate quadrupolar splittings and the relative intensities of multiple Pake patterns underlying each spectrum. For each amantadine orientation, a 1: 3 ratio of two splittings with a 4: 1 ratio of the integrated areas was maintained to represent the 12 equatorial and 3 axial deuterons in the rigid adamantyl cage (Cady et al., 2011b; Cady et al., 2010). The couplings were inputted into EXPRESS to generate Pake patterns. Best fit of the experimental spectra allowed the quantification of the relative intensities of the different drug orientations.

Results Drug-induced chemical shift perturbation to M2(21-97) Fig. 1 compares 2D 15N-13C correlation spectra of three M2 constructs in the absence and presence of amantadine, and also shows the 2D 13C-13C spectra of M2(21-97). Each M2 construct was reconstituted into two lipid membranes, DMPC and VM+. S31 and G34 chemical shifts are diagnostic of the drug-binding status. The TM peptide (Fig. 1a, e) shows a large drug-induced increase of the S31 15N chemical shift in both DMPC and VM+ membranes, indicating drug binding to the pore. In contrast, the intermediate-length TM-AH peptide shows residual S31 intensities at the unbound chemical shifts for the DMPC-bound peptide and no chemical shift change for the VM+ bound peptide (Fig. 1b, f). Consistently, G34 exhibits only partial chemical shift perturbation in the DMPC membrane and no chemical shift perturbation in the VM+ membrane, indicating that amantadine does not bind the pore of the TM-AH peptide in the VM+ membrane. For the cytoplasmic-containing M2(21-97), amantadine caused clear chemical shift changes to the main Ser and Gly cross peaks in both DMPC and VM+ membranes (Fig. 1c, g). These changes are well resolved at the high magnetic fields at which the spectra were measured. Since M2(21-97) is amino- acid-type labeled and contains 8 Ser residues and 4 Gly residues, multiple cross peaks for each residue type are in principle expected. Indeed, the VM+ bound protein shows 3 Ser and 2 Gly N-Cα cross peaks, while the DMPC-bound protein exhibits predominantly one Ser and one Gly N-Cα cross peak. These peaks can be assigned to the transmembrane S31 and G34 based on the chemical shift identity with those of the shorter M2 constructs. We attribute the low peak multiplicity of the amino-acid-type-labeled M2(21-97) to the fact that

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the cytoplasmic tail is highly dynamic and disordered (Liao et al., 2013), thus suppressing Author ManuscriptAuthor Manuscript Author Manuscript Author Manuscript Author most of the intensities of the extra-membrane residues.

The presence of drug-induced chemical shift perturbation in the TM peptide and M2(21-97) while the absence of this perturbation in the TM-AH peptide indicate that the TM peptide reproduces the structural and functional behavior of the fully functional M2, while the intermediate-length peptide does not. The H37 chemical shifts in M2(21-97) further support these observations (Fig. 1d, h), by showing that cationic histidines in the apo samples are converted to neutral histidines in the drug-bound state. At pH 7 for the DMPC sample and pH 8.5 for the VM+ sample, H37 in apo M2(21-97) exhibits three sets of resolved Cα, Cβ and 15N chemical shifts. These peaks have been previously assigned to the neutral tautomers and cationic histidines based on their correlations with the imidazole sidechain chemical shifts and the pH at which these peaks were detected (Liao et al., 2015). For example, the neutral τ tautomer has (Cα, Cβ, N) chemical shifts of (55, 30, 119) ppm, while the π tautomer exhibits an upfield Cβ chemical shift of 28 ppm. The cat0 state has more ideal α- helical (Cα, Cβ) chemical shifts of (60, 30) ppm, while the cat1 chemical shifts can be assigned to a cationic histidine in the +1 charged tetrad (Liao et al., 2015). Importantly, amantadine binding suppressed the cat0 and cat1 signals while retaining the τ and π tautomer peaks, directly indicating that drug binding in the pore prevents protonation of H37 and thus inhibits proton conduction.

Binding site and orientation of the drug associated with M2(21-97) To verify that the chemical shift perturbations seen for the TM-cyto M2 are indeed due to drug binding to the channel pore, we measured 13C-2H REDOR spectra using similar conditions as for the TM and TM-AM peptides (Cady et al., 2011a; Cady et al., 2010). At a molar ratio of 5 drugs per tetramer, the 13C-2H REDOR spectra of VM+ bound M2(21-97) (Fig. 2c) shows clear S31 intensity differences between the control (S0) and dephased (S) spectra, with an S/S0 ratio of 0.77±0.06 at 15.1 ms REDOR mixing. This dephasing value is similar to those of DMPC-bound TM and TM-AH peptides, indicating that the drug binds similarly near S31 in the VM+ bound channels. The dephasing is observed only at S31, while H37 Cα does not show any REDOR dephasing and the overlapped A30 and A29 Cα signal shows a small dephasing, consistent with the local nature of the 13C-2H dipolar coupling.

2 To determine the bound-drug orientation, we measured the static H spectra of d15-Amt in M2(21-97) in DMPC and VM+ membranes. This approach has been used to determine the drug orientation in shorter M2 constructs (Cady et al., 2011b; Cady et al., 2010), and is based on uniaxial rotational diffusion of the drug around its molecular axis and around the bilayer normal. These motions reduce the 2H quadrupolar couplings in an orientation- dependent manner. Among the fifteen C-D bonds, twelve C-D bonds lie at an equatorial orientation of 109° from the molecular axis, while three axial C-D bonds are parallel to the molecular axis. This relation allows us to read off the 2H quadrupolar splittings to determine the orientation of the drug in the lipid bilayer versus inside the channel. The drug orientation is expressed as the tilt angle of the three-fold molecular axis from the bilayer normal. Fig. 3 2 compares eight H spectra of d15-Amt in protein-free and protein-containing DMPC and

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VM+ membranes. As reported before, in protein-free bilayers, amantadine shows a Author ManuscriptAuthor Manuscript Author Manuscript Author Manuscript Author predominant quadrupolar coupling of 18 kHz, indicating a tilt angle of 37° or 80° (Fig. 3a). In TM-containing membranes, a quadrupolar splitting of 36 kHz was observed in both DMPC and VM+ membranes (Fig. 3b), indicating that the drug partitions into the channel pore with a tilt angle of 13°. In TM-AH containing DMPC and VM+ membranes, the 18 kHz splitting became dominant over the 37 kHz splitting, indicating that only a small fraction of drug binds in the pore (Fig. 3c). Finally, in M2(21-97) containing membranes, d15-Amt shows a quadrupolar splitting of 39 kHz for the DMPC sample and 37 kHz in the VM+ sample, which correspond to tilt angles of 2° and 11°, respectively (Fig. 3d). Thus, in the M2(21-97) pore, the drug is vertical, approximately parallel to the bilayer normal. In addition to the 36–39 kHz splitting, we also observed an 11-kHz splitting in the VM+ bound M2(21-97), which we attribute to lipid-bound drug (Table 1).

The amphipathic helix causes membrane curvature To investigate the curvature-inducing effect of M2, we measured static 31P spectra of M2(21-97) in DMPC and VM+ membranes and compared these with previously reported 31P spectra of lipid membranes containing TM and TM-AH peptides (Fig. 4). Membranes containing the TM peptide and TM-cyto M2 show no or relatively weak isotropic peak intensities (Fig. 4a, c), while the TM-AH containing membranes show a strong isotropic peak at 0 ppm, indicating high membrane curvature.

These comparative chemical shift perturbation data, 13C-2H REDOR spectra, and 2H spectra of the drug for the three M2 constructs clarify the complexity of amantadine binding to M2. Drug binding to the TM construct is functional, while the intermediate TM-AH peptide loses the drug-binding ability due to the curvature-inducing function of the amphipathic helix, as seen in the high isotropic peak of the 31P spectra of TM-AH containing membranes. When the full cytoplasmic domain is present, amantadine binding to the pore is restored even when the protein is embedded in the VM+ membrane. Since full-length M2 in the virus lipid envelope is the most biologically relevant condition, the pore-binding site seen in M2(21-97) samples is the functional binding site.

Previous 1H relaxation data and 2D 1H-31P correlation spectra have shown that the intermediate TM-AH M2 not only causes a high-curvature phase but is also partitioned into this phase (Wang and Hong, 2015; Wang et al., 2012). Electron microscopy data showed that the AH’s membrane-curvature induction depends on the cholesterol concentration in the membrane. This M2-cholesterol interaction is complex and is only beginning to be understood. EPR data of spin-labeled M2 indicated that cholesterol reduces the depth of insertion of the AH into the membrane and tightens the inter-helical packing (Kim et al., 2015). 2D 13C-13C correlation NMR spectra showed a cross peak between cholesterol and one of the Phe residues (Ekanayake et al., 2016), giving qualitative evidence that cholesterol is in close contact with M2. Most recently, a higher-resolution structural model of cholesterol bound to TM-AH M2 was determined based on 13C-19F distance measurements, and the bound cholesterol orientation was determined using 2H NMR (Elkins et al., 2017). These data show that cholesterol is bound to the surface of the C-terminal half of the TM domain, with the sterol head near the AH and the isooctyl tail reaching residues in the

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middle of the TM domain. Importantly, only two cholesterol molecules bind each tetramer, Author ManuscriptAuthor Manuscript Author Manuscript Author Manuscript Author thus amplifying the structural asymmetry of the wedge-shaped tetramer (Elkins et al., 2017). We propose that this asymmetric M2-cholesterol complex may alter the TM helix packing such that amantadine can no longer bind to the N-terminal pore. When the cytoplasmic tail is present, the negatively charged residues and the dynamic structure of the tail may attenuate the AH-induced membrane curvature, thus shifting the conformational equilibrium of the TM four-helix bundle towards a state that is competent for drug binding. The cytoplasmic tail may accomplish this by electrostatic repulsion, by changing the AH insertion depth, or by reducing membrane thickening by cholesterol (Cady et al., 2007; Duong-Ly et al., 2005; Saotome et al., 2015).

Membrane-curvature induction by the AH also suggests a potential cause of the recently reported two-fold symmetric dimer-of-dimers structure of the intermediate-length TM-AH M2 (Andreas et al., 2015). The structure was solved in diphytanoylphosphatidylcholine (DPhPC) membranes, which has large negative spontaneous curvature due to the methyl-rich acyl chains (Lindsey et al., 1979). The combination of the intrinsic negative curvature of DPhPC and AH’s curvature-inducing ability may contribute to the breaking of the four-fold symmetry of the channel.

Fig. 5 summarizes the mode of amantadine binding to M2 for all sequence lengths studied to date. Amantadine binds the TM peptide and the fully functional cytoplasmic-containing M2 at the same location in the pore, near S31, and with similar orientations. This binding occurs in both simple phosphocholine membranes and in complex cholesterol-containing membranes. But the drug does not bind the intermediate-length TM-AH M2 in the cholesterol-rich membrane. This loss of binding results from the membrane curvature induced by AH, which is amplified by sub-stoichiometric cholesterol binding to the protein. These two effects together distort the pore geometry of the TM helix bundle. The chemical shifts of S31, G34 and V27 are accurate indicators of the absence or presence of amantadine in the pore, while the chemical shifts of H37 is a reliable reporter of the consequence of drug occlusion of the pore. Therefore, chemical shifts, protein-drug distances, and drug orientations, give a consistent description of the drug binding equilibria and proton-transfer equilibria in this ion channel. Since the TM-AH construct has the same proton conductance as full-length M2 (Ma et al., 2009), the curvature-induction ability of the amphipathic helix is independent of the proton-conduction function of the TM helix. However, the anomalous drug-binding behavior of the TM-AH construct in certain membranes is a manifestation that these two functions intersect through AH-modulated drug binding to the TM pore.

The PIV5 fusion protein: conformational plasticity Enveloped viruses enter cells by merging the viral envelope and the host cell membrane using fusion proteins (Harrison, 2015; White et al., 2008). External events such as receptor binding and pH changes trigger conformational changes of the protein to expose and insert an N-terminal fusion peptide (FP) into the target cell membrane, while a C-terminal transmembrane domain (TMD) remains anchored in the viral envelope (Eckert and Kim, 2001; Kim et al., 2011). From this extended intermediate state, the intervening water-soluble ectodomain undergoes additional dramatic conformational changes that bend itself into a

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hairpin, which forces the FP and TMD into close proximity, in so doing merging the two Author ManuscriptAuthor Manuscript Author Manuscript Author Manuscript Author lipid membranes. This virus-cell fusion model is mainly obtained from crystal structures of the water-soluble ectodomain in the pre-fusion and post-fusion states for a number of viral fusion proteins. For example, for the PIV5 fusion protein F, the prefusion crystal structure shows 11 segments of a hydrophobic heptad repeat region, HRA, within a globular head and a second heptad repeat region, HRB, attached to the bottom of the globular head (Welch et al., 2012; Yin et al., 2006). Upon transition to the post-fusion state, the HRA and HRB assemble into a helical hairpin, which produces a six-helix bundle (6HB) for the trimeric protein (Yin et al., 2005). However, most of these crystal structures lack the membrane- interacting N-terminal FP and C-terminal TMD, thus the structural roles of the FP and TMD in virus-cell fusion are still poorly understood.

Solid-state NMR and solution NMR spectroscopy have been used extensively to investigate the structure and lipid interactions of the fusion peptides of HIV, influenza and parainfluenza virus fusion proteins. These studies showed that membrane compositions, pH, peptide concentrations and construct lengths can exert significant influences on the FP structure. For example, the HIV gp41 FP is α-helical at low peptide concentrations but converts to the β- strand conformation at high peptide concentrations and in cholesterol-containing membranes (Li et al., 2007; Qiang and Weliky, 2009). For influenza hemagglutinin (HA), a 20-residue FP bound to DPC micelles forms a boomerang structure with a bend in the middle, while a 23-residue construct that completes a GxxxG motif forms a tight helical hairpin (Han et al., 2001; Lorieau et al., 2010). In DTPC/DTPG membranes, both the 20-residue and 23-residue FP constructs show a helical hairpin structure but with different interhelical distances (Ghosh et al., 2015). These structural variations strongly suggest that different membrane curvatures can regulate the FP structures.

We have measured the 13C and 15N chemical shifts of the PIV5 fusion protein’s FP (residues 103-129) and TMD (residues 485-510), and found that both peptides have membrane- dependent backbone conformations (Fig. 6a, b) (Yao and Hong, 2014; Yao et al., 2015). The FP adopts an α-helical structure in POPC/POPG membranes but acquires increasing β-sheet content in membranes with spontaneous negative curvature, including DOPC/DOPG, POPE and DOPE membranes. In the most negative-curvature membrane of DOPE, the FP is predominantly βsheet. Both α-helical and β-strand conformations are well inserted into the hydrophobic interior of the membrane. Similar to the FP, the TMD of the protein is α-helical in POPC/cholesterol and POPC/POPG membranes, but shifts to the β-strand conformation in the DOPE membrane. This β-strand conformation is interrupted with an α-helical segment in the center of the peptide, giving a strand-helix-strand motif, indicating that the two termini of the peptide are more sensitive to the membrane curvature.

While studies of isolated FP and TMD peptides give structural information about the early stages of virus-cell fusion, the post-fusion state is known to involve a six-helix-bundle of the ectodomain, which should place the FP and TMD in close proximity in the membrane. We investigated the post-fusion structure of the PIV5 F protein by engineering an FP-TMD chimera, which replaces the 352-residue ectodomain of the PIV5 F protein with a short flexible linker (GGGKKKKK) (Yao et al., 2016). Interestingly, the FP-TMD chimera exhibits α-helical chemical shifts in both lamellar membranes such as POPC/POPG and

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negative-curvature membranes such as DOPE, without a conformational change to β-sheet Author ManuscriptAuthor Manuscript Author Manuscript Author Manuscript Author (Fig. 6c). The fact that tethering the FP and TMD prevents conformational conversion of each domain to the β-sheet structure implies that the inter-domain interactions are stronger than the interaction of each domain with lipids. However, no inter-domain cross peaks between the FP and TMD were detected, suggesting that these two helices do not form an intramolecular hairpin. Solution NMR studies of gp41 in DPC micelles found similar results: the protein exhibits an α-helical conformation but the signals of the C-terminal half residues are severely broadened while the N-terminal signals are narrow, suggesting that the two parts of the protein do not interact strongly with each other and micelle-protein interactions are significant (Lakomek et al., 2014; Lakomek et al., 2013).

Membrane curvature and dehydration by the PIV5 fusion peptide and transmembrane domain The central question in virus-cell fusion is how proteins cause membrane curvature to allow merger of the cell and virus membranes. We investigated the membrane curvature caused by the PIV5 FP and TMD using static 31P NMR (Fig. 7) (Yang et al., 2015; Yao and Hong, 2014; Yao et al., 2016; Yao et al., 2015). These 31P lineshapes indicate that the POPC/POPG membrane retains the lamellar structure in the presence of the FP and TMD, but the DOPE membrane shows an isotropic peak indicative of high curvature upon FP and TMD binding. In particular, the TMD-bound DOPE membrane is quantitatively converted to this high- curvature phase. The isotropic 31P peak can be attributed to micelles, small vesicles, or 31 bicontinuous cubic phases. Small-angle X-ray scattering data (Yao et al., 2015) and P T2 relaxation times (Yang et al., 2015) unambiguously show that this isotropic peak results from a bicontinuous cubic phase, which is rich in the negative Gaussian curvature (NGC) that is geometrically required for the hemifusion and post-fusion states. In addition to causing negative Gaussian curvature, the FP and TMD also dehydrate the membrane surface. This dehydration is observed in 2D 1H-31P correlation spectra that correlate the water and lipid headgroup signals (Yao and Hong, 2014; Yao et al., 2015). In these experiments, the 1H chemical shift of water is encoded in the indirect dimension, then the 1H polarization is transferred during a mixing period to lipids and detected through 31P after cross polarization. These 1H-31P 2D spectra show significantly weakened water cross peak intensities in FP- and TMD-containing PE membranes than for other membranes. The generation of negative Gaussian curvature and membrane dehydration to PE membranes by FP and TMD give strong evidence that these two hydrophobic domains actively participate in membrane fusion, and the β-strand conformation is the relevant conformation that causes the membrane remodeling from the lamellar bilayer to the hemifusion state (Fig. 8).

Compared to the separate FP and TMD peptides, the FP-TMD chimera caused a relatively weak isotropic peak to DOPE (Fig. 7) (Yao et al., 2016), which is consistent with the α- helical conformation of the protein. Interestingly, addition of SDS to DOPE increased the 31 isotropic intensity, and the isotropic peak can be assigned to a cubic phase based on P T2 relaxation times. These results indicate that the FP-TMD chimera causes negative Gaussian curvature to membranes with initial spontaneous positive curvature. The distinct curvature behaviors between the separate peptides and the chimera can be understood in terms of different stages of virus-cell fusion. During membrane fusion, the transition from the

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lamellar bilayer to the hemifusion state exerts negative curvature to the outer leaflet, while Author ManuscriptAuthor Manuscript Author Manuscript Author Manuscript Author the transition from the hemifusion to the post-fusion state incurs positive curvature to the inner leaflet of the membrane. Thus, we attribute the curvature induction by the separate FP and TMD peptides to the formation of the hemifusion intermediate, while the curvature effect of the FP-TMD chimera may be assigned to transition to the post-fusion state. These results are consistent with the biological function of the protein and indicate a fine-tuned interplay between protein structures, lipid-dependent spontaneous membrane curvatures, and the stages of virus-cell fusion.

Conclusion The above review and new experimental data show the complex lipid-protein interactions and protein structural changes involved in two curvature-inducing events of virus budding and virus-cell fusion. The TM helix conformation of the influenza M2 protein is subtly affected by the amphipathic helix, as manifested by the retention or loss of drug in its pharmacologically relevant pore-binding site. The curvature-inducing amphipathic helix, with the help of cholesterol, can perturb the TM helix packing to abolish drug binding, while the cytoplasmic tail restores drug binding. For the PIV5 fusion protein, both the FP and TMD domains exhibit membrane-dependent backbone conformations. By correlating with 31P-NMR detected membrane curvature and water-lipid interactions, we can assign the β- sheet conformation of the isolated peptides to be responsible for the transition from the lamellar bilayer to the hemifusion state, while the transition from the hemifusion state to the post-fusion structure involves α-helical conformation of a FP-TMD chimera that mimics the post-fusion state. Atomic details of this post-fusion structure will require further high- resolution studies of the PIV5 fusion protein and other related viral fusion proteins.

Acknowledgments

This work is funded by NIH grants GM088204 and GM066976 to M.H. The 900 MHz NMR spectra were measured at the MIT/Harvard Center for Magnetic Resonance, which is supported by NIH grant P41-EB-002026.

References Andreas LB, Barnes AB, Corzilius B, Chou JJ, Miller EA, Caporini M, Rosay M, Griffin RG. 2013; Dynamic nuclear polarization study of inhibitor binding to the M2(18-60) proton transporter from influenza A. Biochemistry-Us. 52:2774–2782. Andreas LB, Reese M, Eddy MT, Gelev V, Ni QZ, Miller EA, Emsley L, Pintacuda G, Chou JJ, Griffin RG. 2015; Structure and Mechanism of the Influenza A M218-60 Dimer of Dimers. J Am Chem Soc. 137:14877–14886. [PubMed: 26218479] Cady S, Wang T, Hong M. 2011a; Membrane-dependent effects of a cytoplasmic helix on the structure and drug binding of the influenza virus M2 protein. J Am Chem Soc. 133:11572–11579. [PubMed: 21661724] Cady SD, Mishanina TV, Hong M. 2009a; Structure of amantadine-bound M2 transmembrane peptide of influenza A in lipid bilayers from magic-angle-spinning solid-state NMR: the role of Ser31 in amantadine binding. J Mol Biol. 385:1127–1141. [PubMed: 19061899] Cady SD, Luo WB, Hu FH, Hong M. 2009b; Structure and Function of the Influenza A M2 Proton Channel. Biochemistry-Us. 48:7356–7364. Cady SD, Goodman C, Tatko C, DeGrado WF, Hong M. 2007; Determining the orientation of uniaxially rotating membrane proteins using unoriented samples: a 2H, 13C, and 15N solid-state

J Struct Biol. Author manuscript; available in PMC 2020 April 01. Liao et al. Page 11

NMR investigation of the dynamics and orientation of a transmembrane helical bundle. J Am Chem Author ManuscriptAuthor Manuscript Author Soc. Manuscript Author 129:5719–5729. Manuscript Author [PubMed: 17417850] Cady SD, Wang J, Wu Y, DeGrado WF, Hong M. 2011b; Specific binding of adamantane drugs and direction of their polar amines in the pore of the influenza M2 transmembrane domain in lipid bilayers and dodecylphosphocholine micelles determined by NMR spectroscopy. J Am Chem Soc. 133:4274–4284. [PubMed: 21381693] Cady SD, Schmidt-Rohr K, Wang J, Soto CS, Degrado WF, Hong M. 2010; Structure of the amantadine binding site of influenza M2 proton channels in lipid bilayers. Nature. 463:689–692. [PubMed: 20130653] Duong-Ly KC, Nanda V, Degrado WF, Howard KP. 2005; The conformation of the pore region of the M2 proton channel depends on lipid bilayer environment. Prot Sci. 14:856–861. Eckert DM, Kim PS. 2001; Mechanisms of viral membrane fusion and its inhibition. Annu Rev Biochem. 70:777–810. [PubMed: 11395423] Ekanayake EV, Fu R, Cross TA. 2016; Structural Influences: Cholesterol, Drug, and Proton Binding to Full-Length Influenza A M2 Protein. Biophys J. 110:1391–1399. [PubMed: 27028648] Elkins MR, Williams JK, Gelenter MD, Dai P, Kwon B, Sergeyev IV, Pentelute BL, Hong M. 2017; Cholesterol-binding site of the influenza M2 protein in lipid bilayers from solid-state NMR. Proc Natl Acad Sci U S A. 114:12946–12951. [PubMed: 29158386] Ghosh U, Xie L, Jia LH, Liang S, Weliky DP. 2015; Closed and Semiclosed Interhelical Structures in Membrane vs Closed and Open Structures in Detergent for the Influenza Virus Hemagglutinin Fusion Peptide and Correlation of Hydrophobic Surface Area with Fusion Catalysis. J Am Chem Soc. 137:7548–7551. [PubMed: 26039158] Han X, Bushweller JH, Cafiso DS, Tamm LK. 2001; Membrane structure and fusion-triggering conformational change of the fusion domain from influenza hemagglutinin. Nat Struct Biol. 8:715–720. [PubMed: 11473264] Harrison SC. 2008; Viral membrane fusion. Nature Struc Mol Biol. 15:690–698. Harrison SC. 2015; Viral membrane fusion. Virology. 479:498–507. [PubMed: 25866377] Hong M, DeGrado WF. 2012; Structural basis for proton conduction and inhibition by the influenza M2 protein. Prot Sci. 21:1620–1633. Hu F, Schmidt-Rohr K, Hong M. 2012; NMR detection of pH-dependent histidine-water proton exchange reveals the conduction mechanism of a transmembrane proton channel. J Am Chem Soc. 134:3703–3713. [PubMed: 21974716] Jaroniec CP, Tounge BA, Herzfeld J, Griffin RG. 2001; Frequency Selective Heteronuclear Dipolar Recoupling in Rotating Solids: Accurate 13C-15N Distance Measurements in Uniformly 13C, 15N-labeled Peptides. J Am Chem Soc. 123:3507–3519. [PubMed: 11472123] Kim SS, Upshur MA, Saotome K, Sahu ID, McCarrick RM, Feix JB, Lorigan GA, Howard KP. 2015; Cholesterol-Dependent Conformational Exchange of the C-Terminal Domain of the Influenza A M2 Protein. Biochemistry-Us. 54:7157–7167. Kim YH, Donald JE, Grigoryan G, Leser GP, Fadeev AY, Lamb RA, DeGrado WF. 2011; Capture and imaging of a prehairpin fusion intermediate of the paramyxovirus PIV5. Proc Natl Acad Sci USA. 108:20992–20997. [PubMed: 22178759] Kwon B, Tietze D, White PB, Liao SY, Hong M. 2015; Chemical ligation of the influenza M2 protein for solid-state NMR characterization of the cytoplasmic domain. Prot Sci. 24:1087–1099. Lakomek NA, Kaufman JD, Stah SJ, Wingfield PT. 2014; HIV-1 Envelope Protein gp41: An NMR Study of Dodecyl Phosphocholine Embedded gp41 Reveals a Dynamic Prefusion Intermediate Conformation. Structure. 22:1311–1321. [PubMed: 25132083] Lakomek NA, Kaufman JD, Stahl SJ, Louis JM, Grishaev A, Wingfield PT, Bax A. 2013; Internal Dynamics of the Homotrimeric HIV-1 Viral Coat Protein gp41 on Multiple Time Scales. Angew Chem Int Ed Engl. 52:3911–3915. [PubMed: 23450638] Lamb RA, Jardetzky TS. 2007; Structural basis of viral invasion: lessons from paramyxovirus F. Curr Opin Struct Biol. 17:427–436. [PubMed: 17870467] Li C, Qin H, Gao FP, Cross TA. 2007; Solid-state NMR characterization of conformational plasticity within the transmembrane domain of the influenza A M2 proton channel. Biochim Biophys Acta. 1768:3162–3170. [PubMed: 17936720]

J Struct Biol. Author manuscript; available in PMC 2020 April 01. Liao et al. Page 12

Liao SY, Fritzsching KJ, Hong M. 2013; Conformational analysis of the full-length M2 protein of the Author ManuscriptAuthor Manuscript Author Manuscriptinfluenza Author A virus using Manuscript Author solid-state NMR. Prot Sci. 22:1623–1638. Liao SY, Yang Y, Tietze D, Hong M. 2015; The influenza m2 cytoplasmic tail changes the proton- exchange equilibria and the backbone conformation of the transmembrane histidine residue to facilitate proton conduction. J Am Chem Soc. 137:6067–6077. [PubMed: 25892574] Lindsey H, Petersen NO, Chan SI. 1979; Physicochemical characterization of 1,2-diphytanoyl-sn- glycero-3-phosphocholine in model membrane systems. Biochim Biophys Acta. 555:147–167. [PubMed: 476096] Lorieau JL, Louis JM, Bax A. 2010; The complete influenza hemagglutinin fusion domain adopts a tight helical hairpin arrangement at the lipid:water interface. Proc Natl Acad Sci USA. 107:11341– 11346. [PubMed: 20534508] Luo W, Hong M. 2010; Conformational changes of an ion channel membrane protein detected through water-protein interactions using solid-state NMR spectroscopy. J Am Chem Soc. 132:2378–2384. [PubMed: 20112896] Luo W, Cady SD, Hong M. 2009; Immobilization of the Influenza A M2 Transmembrane Peptide in Virus-Envelope Mimetic Lipid Membranes: A Solid-State NMR Investigation. Biochemistry-Us. 48:6361–6368. Ma C, Polishchuk AL, Ohigashi Y, Stouffer AL, Schon A, Magavern E, Jing X, Lear JD, Freire E, Lamb RA, DeGrado WF, Pinto LH. 2009; Identification of the functional core of the influenza A virus A/M2 proton-selective ion channel. Proc Natl Acad Sci USA. 106:12283–12288. [PubMed: 19590009] McMahon HT, Gallop JL. 2005; Membrane curvature and mechanisms of dynamic cell membrane remodelling. Nature. 438:590–596. [PubMed: 16319878] Pinto LH, Lamb RA. 2006; The M2 proton channels of influenza A and B viruses. J Biol Chem. 281:8997–9000. [PubMed: 16407184] Qiang W, Weliky DP. 2009; HIV Fusion Peptide and Its Cross-Linked Oligomers: Efficient Syntheses, Significance of the Trimer in Fusion Activity, Correlation of beta Strand Conformation with Membrane Cholesterol, and Proximity to Lipid Headgroups. Biochemistry-Us. 48:289–301. Rossman JS, Lamb RA. 2011; Influenza virus assembly and budding. Virology. 411:229–236. [PubMed: 21237476] Rossman JS, Jing X, Leser GP, Lamb RA. 2010; Influenza virus M2 protein mediates ESCRT- independent membrane scission. Cell. 142:902–913. [PubMed: 20850012] Sack II, Goldbourt A, Vega S, Buntkowsky G. 1999; Deuterium REDOR: principles and applications for distance measurements. J Magn Reson. 138:54–65. [PubMed: 10329226] Saotome K, Duong-Ly KC, Howard KP. 2015; Influenza A M2 protein conformation depends on choice of model membrane. Biopolymers. 104:405–411. [PubMed: 25652904] Schnell JR, Chou JJ. 2008; Structure and mechanism of the M2 proton channel of influenza A virus. Nature. 451:591–595. [PubMed: 18235503] Sinha N, Schmidt-Rohr K, Hong M. 2004; Compensation for Pulse Imperfections in Rotational-Echo Double-Resonance NMR by Composite Pulses and EXORCYCLE. J Magn Reson. 168:358–365. [PubMed: 15140448] Stouffer AL, Acharya R, Salom D, Levine AS, Di Costanzo L, Soto CS, Tereshko V, Nanda V, Stayrook S, DeGrado WF. 2008; Structural basis for the function and inhibition of an influenza virus proton channel. Nature. 451:596–599. [PubMed: 18235504] Vold RL, Hoatson GL. 2009; Effects of jump dynamics on solid state nuclear magnetic resonance line shapes and spin relaxation times. J Magn Reson. 198:57–72. [PubMed: 19201232] Wang T, Hong M. 2015; Investigation of the Curvature Induction and Membrane Localization of the Influenza Virus M2 Protein Using Static and Off-Magic-Angle-Spinning Solid-State Nuclear Magnetic Resonance of Oriented Bicelles. Biochemistry-Us. 54:2214–2226. Wang T, Cady SD, Hong M. 2012; NMR Determination of Protein Partitioning into Membrane Domains with Different Curvatures and Application to the Influenza M2 Peptide. Biophys J. 102:787–794. [PubMed: 22385849]

J Struct Biol. Author manuscript; available in PMC 2020 April 01. Liao et al. Page 13

Welch BD, Liu Y, Kors CA, Leser GP, Jardetzky TS, Lamb RA. 2012; Structure of the cleavage- Author ManuscriptAuthor Manuscript Author Manuscriptactivated Author prefusion form Manuscript Author of the parainfluenza virus 5 fusion protein. Proc Natl Acad Sci U S A. 109:16672–16677. [PubMed: 23012473] White JM, Delos SE, Brecher M, Schornberg K. 2008; Structures and mechanisms of viral membrane fusion proteins: Multiple variations on a common theme. Crit Rev Biochem Mol Biol. 43:189– 219. [PubMed: 18568847] Williams JK, Tietze D, Wang J, Wu Y, DeGrado WF, Hong M. 2013; Drug-induced conformational and dynamical changes of the S31N mutant of the influenza M2 proton channel investigated by solid-state NMR. J Am Chem Soc. 135:9885–9897. [PubMed: 23758317] Yang Y, Yao HW, Hong M. 2015; Distinguishing Bicontinuous Lipid Cubic Phases from Isotropic Membrane Morphologies Using P-31 Solid-State NMR Spectroscopy. J Phus Chem B. 119:4993– 5001. Yao HW, Hong M. 2014; Conformation and Lipid Interaction of the Fusion Peptide of the Paramyxovirus PIV5 in Anionic and Negative-Curvature Membranes from Solid-State NMR. J Am Chem Soc. 136:2611–2624. [PubMed: 24428385] Yao HW, Lee M, Liao SY, Hong M. 2016; Solid-State Nuclear Magnetic Resonance Investigation of the Structural Topology and Lipid Interactions of a Viral Fusion Protein Chimera Containing the Fusion Peptide and Transmembrane Domain. Biochemistry-Us. 55:6787–6800. Yao HW, Lee MW, Waring AJ, Wong GCL, Hong M. 2015; Viral fusion protein transmembrane domain adopts beta-strand structure to facilitate membrane topological changes for virus-cell fusion. Proc Natl Acad Sci USA. 112:10926–10931. [PubMed: 26283363] Yin HS, Paterson RG, Wen X, Lamb RA, Jardetzky TS. 2005; Structure of the uncleaved ectodomain of the paramyxovirus (hPIV3) fusion protein. Proc Natl Acad Sci U S A. 102:9288–9293. [PubMed: 15964978] Yin HS, Wen X, Paterson RG, Lamb RA, Jardetzky TS. 2006; Structure of the parainfluenza virus 5 F protein in its metastable, prefusion conformation. Nature. 439:38–44. [PubMed: 16397490]

J Struct Biol. Author manuscript; available in PMC 2020 April 01. Liao et al. Page 14 Author ManuscriptAuthor Manuscript Author Manuscript Author Manuscript Author

Figure 1. 2D 15N-13C and 13C-13C correlation spectra of three M2 constructs in DMPC (a-d) and VM + (e-h) membranes without and with drug. (a, e) The TM peptide (residues 22-46). (b, f) The intermediate TM-AH peptide (residues 21-61). (c, g) The cytoplasmic-containing TM-cyto construct (residues 21-97). (d, h) H37 region of the 13C-13C correlation spectra of TM-cyto. Apo spectra are shown in black and drug-bound spectra are shown in red. The drug: tetramer molar ratio is 5: 1. The spectra were measured at 400 MHz for the two shorter peptides and at 800 and 900 MHz 1H Larmor frequencies for M2(21-97), thus the latter spectra have higher resolution. VM+ bound TM-AH M2 has no chemical shift perturbations by the drug, while VM+ bound TM and TM-cyto show clear drug-induced chemical shift perturbations at S31, G34, and H37.

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Figure 2. 13 2 C- H REDOR spectra (S0, S and difference spectra) of three M2 constructs. (a) DMPC- bound TM peptide. (b) DMPC-bound TM-AH peptide. (c) VM+-bound TM-cyto M2. d15- Amt was added to the samples at 4:1 or 5:1 drug: tetramer ratios. Clear 13C-2H dipolar dephasing was observed for S31 in all three samples, indicating that Amt binds to the N- terminal pore of the channel. Notably, this occurs for the cytoplasmic-containing M2 when reconstituted into the VM+ membrane, indicating that this complex membrane does not perturb drug binding to the TM-cyto protein.

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Figure 3. 2 Static H spectra of d15-Amt in DMPC (top row) and VM+ (bottom row) membranes with and without M2. The spectra were measured at 303 K. The drug: tetramer ratios are indicated. (a) Amantadine in DMPC and VM+ membranes without M2. (b) Amantadine bound to TM-containing membranes. (c) Amantadine bound to TM-AH containing membranes. (d) Amantadine bound to TM-cyto containing membranes. Simulated 2H spectra (red) are superimposed with the experimental spectra (black). The 11-kHz splitting in the (d) has not been observed in the shorter M2 constructs. Assignment notations in the brackets are: L: lipid; P: pore; E: equatorial; A: axial; 0°: 0° edges of the Pake pattern; 90°: 90° edges of the Pake pattern. Tilt angles of the drug from the bilayer normal are indicated for various quadrupolar splittings.

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Figure 4. Static 31P spectra of DMPC (top row) and VM+ (bottom row) membranes containing the three M2 constructs. Drug: tetramer molar ratios are indicated for each sample. (a) Membranes containing the TM peptide. (b) Membranes containing the TM-AH peptide. (c) Membranes containing the TM-cyto protein. The TM-AH M2 causes a strong 31P isotropic peak to both DMPC and VM+ membranes, indicating high curvature. The TM-cyto samples do not show an isotropic peak, indicating that the cytoplasmic tail attenuates the curvature- inducing effect of the AH.

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Figure 5. Illustration of drug binding to M2 as a function of the sequence length. (a) Amantadine binds to the channel pore of the TM peptide in all membranes studied. (b) Amantadine does not bind the intermediate TM-AH M2 when the peptide is in the cholesterol-rich virus- mimetic membrane but partially binds the channel pore when the peptide is embedded in the DMPC membrane. (c) Drug binds the channel pore of cytoplasmic-containing M2 in both DMPC and VM+ membranes. (d) Amantadine has a tilt angle of 2° in the DMPC-bound TM-cyto M2 and 11° in the VM+ bound TM-cyto M2.

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Figure 6. Representative 2D 13C-13C correlation spectra of the PIV5 fusion protein domains demonstrate the influence of the membrane composition on protein conformation. Left: POPC/POPG membrane. Right: DOPE membrane. The spectra were measured between −30°C and −10°C. (a) FP. (b) TMD. (c) FP-TMD chimera. α-helical and β-sheet resonances are assigned in red and blue, respectively. FP and TMD peptides show more β-sheet peaks in the DOPE membrane than in the POPC/POPG membrane, while the FP-TMD chimera shows stable α-helical conformation in both membranes. Unassigned peaks in (c) come from a fusion tag.

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Figure 7. 31P NMR spectra detecting the effects of the PIV5 fusion protein domains on membrane curvature. (a) POPC/POPG membranes. (b) DOPE membranes. Spectra in red were measured on membranes containing the FP (top row), TMD (middle row), and the FP-TMD chimera (bottom row). The spectra were measured at 30°C for FP- and TMD-containing membranes and 25°C for FP-TMD containing samples. These 31P lineshapes indicate that the peptides do not perturb the lamellar structure of the POPC/POPG membrane, but induce varying intensities of an isotropic peak to the DOPE membrane. The isotropic peak represents 15%, 75% and 12% of the total spectral intensities of the DOPE membranes containing the FP, TMD, and FP-TMD, respectively.

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Figure 8. Structural model of the PIV5 fusion protein FP and TMD in PE-rich membranes. The FP mainly adopts a β-strand conformation while the TMD has a strand-helix-strand structure. These β-strand-rich conformations cause negative Gaussian curvature to the PE membrane, which is essential for hemifusion intermediates during membrane fusion.

J Struct Biol. Author manuscript; available in PMC 2020 April 01. Liao et al. Page 22 Author ManuscriptAuthor Manuscript Author Manuscript Author Manuscript Author . Where two quadrupolar splittings are listed, the first value is that of the 12 δ Table 1 -Amt in lipid membranes without and with M2(21-97). 5% 8% 4% 95% 78% 10% 15 Fraction a VM+ 0 0 37 11 (kHz) 19, 60 19, 63 Q Δv - 2% 98% 80% 10% 10% Fraction a ) and intensity fractions of d DMPC Q - 0 0 39 (kHz) 18, 59 20, 64 Q Δv Pore Lipid Lipid Surface Isotropic Isotropic Binding site b No M2 Samples With M2 H quadrupolar splittings (Δv The drug: tetramer molar ratio is 5: 1. All quadrupolar splittings are read off from the 90° edges of Pake patterns and thus correspond to the anisotropy parameter 2 a equatorial C-D bonds while the second is that of 3 axial bonds. Where only one quadrupolar splitting listed, value is that of the equatorial C-D bonds. b

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