Membrane interaction and structure of the transmembrane domain of influenza hemagglutinin and its fusion peptide complex
© Chang et al; licensee BioMed Central Ltd. 2008
Received: 29 November 2007
Accepted: 15 January 2008
Published: 15 January 2008
To study the organization and interaction with the fusion domain (or fusion peptide, FP) of the transmembrane domain (TMD) of influenza virus envelope glycoprotein for its role in membrane fusion which is also essential in the cellular trafficking of biomolecules and sperm-egg fusion.
The fluorescence and gel electrophoresis experiments revealed a tight self-assembly of TMD in the model membrane. A weak but non-random interaction between TMD and FP in the membrane was found. In the complex, the central TMD oligomer was packed by FP in an antiparallel fashion. FP insertion into the membrane was altered by binding to TMD. An infrared study exhibited an enhanced membrane perturbation by the complex formation. A model was built to illustrate the role of TMD in the late stages of influenza virus-mediated membrane fusion reaction.
The TMD oligomer anchors the fusion protein in the membrane with minimal destabilization to the membrane. Upon associating with FP, the complex exerts a synergistic effect on the membrane perturbation. This effect is likely to contribute to the complete membrane fusion during the late phase of fusion protein-induced fusion cascade. The results presented in the work characterize the nature of the interaction of TMD with the membrane and TMD in a complex with FP in the steps leading to pore initiation and dilation during virus-induced fusion. Our data and proposed fusion model highlight the key role of TMD-FP interaction and have implications on the fusion reaction mediated by other type I viral fusion proteins. Understanding the molecular mechanism of membrane fusion may assist in the design of anti-viral drugs.
Influenza hemagglutinin (HA) is responsible for the attachment and fusion of the virus to the target membrane. Mature HA is composed of HA1 (attachment) and HA2 (fusion) subunits connected by a disulfide linkage. HA2 can be divided into the fusion peptide (FP) domain, the heptad repeat (HR) regions, transmembrane domain (TMD) and the cytoplasmic tail (CT). The functional roles of FP and HR domains have been demonstrated rather clearly [1–4]: the hydrophobic FP domain is sequestered in the resting state but exposed and inserted into the target membrane on low pH activation; the HR domain undergoes extensive refolding to form the hairpin structure to bring the two membranes proximal and probably provides free energy to overcome the barrier of membrane merger. A previous study by Lai et al.  revealed that the functional fusion peptide of influenza virus had a kinked helix structure with a fixed angle in the micellar environment. However, the role played by TMD remains controversial except for the recognition that it anchors the fusion protein on the viral membrane and is involved in the late stages of the fusion process. As evidence for the latter proposition, cells expressing a glycosylphosphatidylinositol (GPI)-anchored ectodomain of HA have been shown to support hemifusion to target membranes at low pH , implying a TMD role in transiting membrane hemifusion to full fusion. The result was corroborated by a stringent TMD length requirement for supporting full membrane fusion , strongly suggesting that it is necessary for TMD to span both inner and outer leaflets to fulfill its function of driving complete fusion via hemifusion. On the other hand, a mutational study of the HIV-1 TMD demonstrated that substitution of one specific residue in TMD did not alter the fusion protein function, whereas replacement of TMD with that of CD4  or of vesicular stomatitis virus G  abolished the viral fusion activity without affecting transport and cleavage properties.
The structure, orientation and interaction of the TMD of HA2 (X:31 strain) has been investigated by Tatulian and Tamm . It was found that the highly helical TMD inserted into lipid bilayer nearly perpendicular to the membrane surface, probably forming oligomers of various sizes and water-accessible pores. They suggested that TMD had a role at the late stages of membrane fusion, including dehydration of water at the apposing membrane surfaces. Melikyan et al.  have shown that substitution of the TMD of HA (Japan) with TMD from other unrelated proteins does not affect membrane fusion. On the other hand, mutation of selected residues within TMD abolished fusion .
As a widely held model on protein-induced fusion proposes that the ectodomain of fusion proteins consists of heptad repeat domains sandwiched between FP and TMD capable of forming a helix hairpin, it is of interest to explore whether there exists any interaction between FP and TMD and, if so, what is the nature of the interaction and its involvement in the fusion process. In addition, to clarify the architecture of TMD in the membrane in complex with FP, we conducted biophysical experiments on the peptides derived from HA2 TMD and FP in a model membrane. Owing to the potentially weak interaction between TMD and FP in the membranous environment, fluorescence spectrophotometry was employed which is most suitable for long-range (>10 Å) interactions in lieu of the nuclear magnetic resonance (NMR) measurements that are sensitive to short-range association (<5 Å). We found that TMD self-associated more tightly than FP in the membrane. The two peptide molecules form a loose complex in an antiparallel manner, with TMD oligomers interspersed with FP molecules and modulation of lipid penetration of FP by interacting with TMD.
An operational model of the fusion mechanism based on the findings of the present work and previous study was constructed to shed light on the role of TMD and FP with an emphasis on the promotion of the transition from hemifusion to full fusion by the two regions in HA2 represented by TMD and FP.
Elucidation of the function of TMD and FP and their interaction in the context of HA-induced membrane fusion may provide a missing piece in the mechanistic study of a host of cell-cell and cell-virus fusion events in which the helix-bundle was shown to be the core structure, for example, in fusion mediated by other proteins involved in the intracellular vesicle fusion [13–15].
Influenza TMD peptide associates with and inserts into the membrane
Self-assembly of TMD in the membrane bilayer can be deduced from Rhodamine self-quenching by variation of composition of the fluorescent-labeled peptide
The nature of binding between TMD and FP was revealed in Figure 2B. For both pH levels tested, the normalized intensity of the Rho dye labeled to TMD or FP of HA2 increases on mixing with their counterparts, while no change is observed when FP of human immunodeficiency virus is added to the TMD-containing solution. This result clearly indicates that the interaction between TMD and FP is not random. We also found that the intensity enhancement is less pronounced for the labeled TMD than the labeled FP when the TMD:FP complex is formed, indicating that self-packing of TMD is tighter and TMD likely forms the inner core in the complex. The difference is easily detected in top and middle panels of Figure 2B for pH 7.4 in which Rho-TMD experiences less dequenching than Rho-FP at the same x value; also, at smaller x, Rho-FP shows a larger increase suggesting a dispersed FP subunit by complexing to TMD (i.e. reduced intra-subunit association), supporting the concept of an inner TMD core for the TMD:FP complex which is more directly shown in the next section.
Rhodamine self-quenching measurements reveal association of TMD with FP and TMD probably forms the inner core in the membrane
Assembly of TMD and TMD:FP complex of HA as probed by the Rhodamine self-quenching. The FP peptide of HIV gp41 was used as a negative control. Values are expressed as a percentage of the Rhodamine intensity in the presence of 0.2% Triton X-100.
It is noteworthy that a substantial Rho-TMD dequenching upon addition of FP is observed in the composition variation study, particularly at low x values (Figure 2B) while little dequenching is found for Rho-TMD complexed to FP. Conceivably, the long-range interaction between the fluorescent labels attached to TMD, which is monitored in the low x regime (Figure 2B), is affected by FP addition; however, the short-range interaction probed by experiments leading to data in Table 1 using fully labeled TMD exhibits little change with FP addition, indicating a very compact TMD oligomer (possibly trimer) subunit un-dissociable by complexing to FP.
Again the association for both FP and TMD in the complex is tighter at acidic pH than neutral pH (Table 1).
FRET measurements between NBD and Rhodamine afford evidence for interaction between TMD and FP
FP molecules are arranged in antiparallel orientation in the TMD:FP complex
Insertion depth of HA2 FP is altered by the interaction with TMD
The Tb3+/DPA measurements suggest that the HA2 TMD peptide does not exhibit membrane leakage activity as FP does
HA2 TMD inserts into membrane nearly perpendicularly and promotes dehydration but causes less membrane perturbation than FP as revealed by ATR-FTIR measurements
The secondary structure and orientation of helix, beta sheet and lipid acyl chain of FP, TMD and FP/TMD 1:1 complex in DMPC:DMPG 1:1 vesicular solution with L/P = 50 at pH 5.0. Values were obtained by averaging three or four sets of data.
26 ± 3
64 ± 1
46 ± 7
55 ± 5
20 ± 2
28 ± 1
9 ± 3
9 ± 8
11 ± 3
17 ± 4
17 ± 3
Helix axis orientation
1.82 ± 0.01
2.46 ± 0.22
2.07 ± 0.08
60 ± 1
34 ± 1
49 ± 2
1.18 ± 0.10
1.28 ± 0.20
56 ± 5
52 ± 7
Acyl chain tilt angle
1.09 ± 0.09
1.78 ± 0.45
1.31 ± 0.04
1.62 ± 0.12
27 ± 4
49 ± 12
36 ± 1
45 ± 4
TMD of HA2 inserts into membrane bilayer with a pH-dependent depth
We have shown that HA2 FP penetrated more deeply into the membrane at low pH. The result in Figure 1 on TMD membrane-insertion depth displays similar pH dependence. The deeper insertion at acidic pH for both TMD and FP, as discussed in the following, may have ramifications for the low-pH activation of HA2-mediated fusion process.
Self-assembly of TMD is stronger than FP and is insignificantly affected by the incorporation of FP
A previous investigation revealed loose self-association of FP in the membrane . Here we show in Table 1 that TMD molecules form tightly packed oligomeric subunits in the membrane which are tighter than FP as deduced from the greater Rhodamine self-quenching for TMD. No discernible dequenching is observed for Rhodamine-labeled TMD as FP is added, while Rhodamine conjugated to FP has enhanced dequenching with TMD incorporation. This suggests that tight TMD packing is intact upon interacting with FP whereas inter-FP distance becomes longer for loosely aggregated FP monomers when attracted by tightly associated TMD oligomers nearby. Another line of evidence for a more stable oligomer formed by TMD can be visualized in Figure 2C, in which only the monomeric FP band is displayed. More indirect evidence for tighter association of TMD than FP and that TMD constitutes the inner core of the TMD:FP complex can be deduced from Figure 2B. The association between the two kinds of molecules is further affirmed by the FRET results shown in Figure 3 indicating larger transfer efficiency than random distribution of the two peptides from NBD to Rhodamine conjugated, respectively, to TMD and FP at the opposite ends. The orientation between TMD and FP can be resolved by FRET experiments in which pyrene (donor) and NBD (acceptor) were labeled to TMD and FP at either N- or C-terminus (Figure 4). The result clearly showed an antiparallel TMD:FP association.
Membrane interaction of TMD and FP
It is interesting that, unlike FP, TMD displays little membrane disrupting effect despite the closer TMD packing, as shown in the leakage experiments summarized in Figures 6A and 6B. This is corroborated by the ATR-FTIR data on the dehydration (Figure 7) and lipid acyl chain orientation (Table 2). This could be explained by the smaller membrane insertion angle (closer to membrane normal) for TMD, causing less membrane perturbation. After all, TMD serves as an anchor for the viral fusion protein and therefore should not induce membrane permeation and death of the virus.
The membrane perturbing effect of TMD and FP has also been studied by ATR-IR measurements as shown in Figure 7. The larger fraction of carbonyl vibrational peak for the TMD:FP complex than that for either TMD or FP reveals a synergistic membrane-perturbing effect of the TMD:FP complex. As membrane dehydration represents a major barrier to fusion, this result suggests that association of the two HA2 domains, primarily by perturbing the membrane bilayer at the fusing site, promotes membrane merger mediated by the influenza hemagglutinin.
In this work, fluorophotometry, such as FRET and Rhodamine self-quenching, was used to study the association between TMD and FP and membrane organization of TMD. It turns out that this is appropriate because the active distance for these fluorescence measurements is in the range of 10–50 Å, which covers the loose interaction between FP and TMD. The loose TMD:FP complex inferred from the present work is in line with the sodium dodecyl sulfate gel electrophoresis experiment in which the two coincubated peptides exhibited separate TMD and FP bands under the electric field and dispersing force of SDS (Figure 2C).
Biological implication of FP:TMD interaction
We have provided several lines of evidence for the loose association between TMD and FP in the model membrane, in contrast to highly specific recognition of the receptor by the surface subunit of the viral fusion protein. Perhaps the role of TMD in the membrane fusion is twofold: first, mechanically it anchors the fusion protein onto the viral membrane and secures the oligomerization of the fusion protein through its tight self-association, and importantly, it does not destabilize the membrane in the absence of FP; second, it has a weak interaction with FP, thereby reinforcing the destabilizing effect of FP on the inner leaflet of the target membrane by deepening FP membrane insertion (Figure 7). This latter effect is manifested by the requirement of TMD length for different phenotypes of fusion, hemifusion and full-fusion activity , because spanning both leaflets of the bilayer for TMD is conceivably a prerequisite for TMD to execute this function. The differential results on the effect of altering the basic residue in the middle of the HIV-1 TMD sequence [9, 24] may be related to the weak association between TMD and FP deduced herein (Figure 2C). The concept that the role of TMD in the fusion process lies more in disrupting the inner leaflet of the fusing membranes than the specific interaction with FP is consistent with the inability of a GPI-anchored HA ectodomain to mediate full fusion .
The results presented in the work highlight the importance of the interaction of TMD with the membrane and TMD in complex with FP in the steps leading to pore initiation and dilation and shed some light on the fusion reaction mediated by other type I viral fusion proteins.
The DMPC, DMPG and POPC used in this work were obtained from Avanti Polar Lipids (Alabaster, AL, USA), acrylamide, NBD and Triton X-100 from Sigma (St. Louis, MO, USA) and 5(6)-carboxytetramethylrhodamine hydrochloride (TAMRA) from Molecular Probes, Inc. (MPI, Eugene, OR, USA). Terbium chloride hexahydrate (TbCl3) and 2,6-pyridinedicarboxylic acid (DPA) were purchased from Acros Organics (Geel, Belgium). All reagents were used without further purification.
The peptides of TMD (GYKDWILWISFAISCFLLCVVLLGFIMWACQRG) and FP (GLFGAIAGFIENGWEGMIDGWYGFR) of HA2 (strain X:31) of influenza virus were synthesized using a Fmoc/t-Bu solid-phase method on a Rainin PS3 peptide synthesizer (Protein Technologies, Tucson, AZ, USA). Labeling of TAMRA or NBD, purification and characterization of the peptides used were described previously [25, 26]. The pyrene labeling protocol was detailed in Additional file 1. Lys was added at the end of the sequence while the fluorescent probes were labeled on the C-terminus.
Small unilamellar vesicles (SUVs) were prepared by solubilizing DMPC:DMPG mixture (1:1) in chloroform:methanol (4:1, v/v). The lipidic solution was dried under a stream of nitrogen until a thin film was obtained and then dried using a centrifuge under vacuum overnight to ensure the movement of all solvent. The phospholipid was resuspended in PB buffer and sonicated for 30 min with a Sonicor (New York, NY, USA) ultrasonic processor.
All fluorescence experiments were performed on a Hitachi F-2500 Fluorescence Spectrophotometer at 37°C, unless indicated otherwise. A scan rate of 300 nm/min was used in the wavelength scan measurements.
Acrylamide quenching experiments
The fluorescence quenching study monitors the accessibility of Trp to the acrylamide quencher. Thus, a larger quenching constant of Trp by the aqueous phase quencher acrylamide indicates that the Trp is located closer to the membrane interface. Fluorescence emission spectra in the 300–450 nm range were recorded by using a 280 nm excitation wavelength with a cutoff filter at 300 nm. The slit bandwidths of excitation and emission were 5 and 2.5 nm, respectively. An incremental amount of acrylamide stock solution (1 M) was added to the 1 μM TMD peptide solutions (in PB buffer or in DMPC:DMPG 50:50 μM) to make final concentration of acrylamide up to 50 mM. Appropriate blanks were subtracted to obtain the corrected spectra and corrections owing to dilution were made to the observed fluorescence intensities. The data were analyzed using the Stern-Volmer equation :
F 0/F = 1 + K SV·[Q] (1)
where F 0 is the fluorescence intensity at the zero quencher concentration, F is the fluorescence intensity at any given quencher concentration [Q], whereas K SV represents the apparent Stern-Volmer quenching constant, obtained from the slope of the plot of F 0/F versus [Q].
Rho-labeled/unlabeled peptide composition experiments
In the experiments on the composition variation of Rho-labeled peptide, the fraction of labeled peptide, x, was varied from 0.02 or 0.05 to 1. For self-association measurements of HA2 TMD or FP, the concentrations were kept at 1 μM/100 μM/100 μM of peptide/DMPC/DMPG. To investigate the association between TMD and FP of HA2 or HIV, a total concentration of 0.06 μM of each peptide (labeled and unlabeled) in DMPC:DMPG 30 μM:30 μM was used. Excitation and emission wavelengths of 530 and 578 nm, respectively, were used with slit bandwidth of excitation and emission of 10 nm. The normalized emission intensity I x /x was plotted against 1 - x .
It is noted that intra-trimeric interaction is detected for x values near 1 since nearly all peptide molecules are labeled and, therefore, quenching arises predominantly from the close neighbors within the same trimer. In contrast, for low x values, the probability of finding a pair of labeled peptides is slim and hence quenching arises mainly from labeled peptides in nearby trimers.
Association tendency of TMD and FP by Rho fluorophore
The Rho self-quenching experiments were carried out to examine the propensity of association of TMD with FP. To DMPC:DMPG (30/30 μM) vesicles at pH 5.0 or 7.4, the Rho-labeled TMD (or FP) was added followed by adding the unlabeled FP (or TMD). We used 0.06 μM of each peptide and the parameters were the same as those used in the Rho composition experiments described above. The 100% reference intensity was taken from the fluorescence measured in the peptide/lipid dispersion solubilized with 0.2% (v/v) Triton X-100.
FRET between Rho-labeled TMD peptide and NBD-labeled FP
The Förster distance (R 0), at which the FRET efficiency is 50%, of the NBD-Rho pair (donor-acceptor) is about 56 Å . NBD and Rho were labeled on FP and TMD peptides, respectively, at either N- or C-terminal end. The FRET between NBD and Rho was measured at 50°C by adding Rho-TMD to NBD-FP/DMPC/DMPG 0.06:150:150 μM. The ratios of [Rho-TMD]/[NBD-FP] were 0.3, 0.6, 1, 1.5, 2 and 2.5. To investigate the changes of NBD intensity, the excitation and the emission wavelengths were set at 467 and 530 nm, respectively, with a response of 0.04 s and slit bandwidth of excitation and emission of 10 nm.
To calculate the FRET efficiency, the intensity of donor (NBD-FP) without acceptor (Rho-TMD) was taken as 100%:
Efficiency (%) = I donor+acceptor/I donor × 100 (2)
where I donor+acceptor and I donor are the intensities of NBD-FP/Rho-TMD mixture and NBD-FP only, respectively.
FRET between Pyrene-labeled TMD peptide and NBD-labeled FP
The measurements of FRET from Pyrene to NBD were recorded to investigate the alignment between TMD and FP peptides. The Förster distance R 0 of the pyrene-NBD pair (donor-acceptor) is about 33 Å . TMD and FP peptides were labeled by pyrene and NBD, respectively, on either N-terminus or C-terminus. Pyrene-labeled TMD was added to the DMPC:DMPG (15:15 μM) vesicular solution followed by the addition of the same amount of NBD-labeled FP. The final concentration of each peptide was 0.06 μM. To monitor the pyrene probe, the excitation and the emission wavelengths were set at 344 and 380 nm, respectively, with slit bandwidth of excitation and emission of 10 nm.
FRET efficiency is calculated according to (2) except that I donor+acceptor and I donor are the intensities of pyrene-TMD/NBD-FP mixture and pyrene-TMD only, respectively.
NBD fluorescence can be quenched by divalent cobalt ions  via a collisional quenching mechanism. Similar to acrylamide quenching of Trp, a large quenching constant by the aqueous cation reflects a closer proximity of NBD tag to the membrane interface. For Co2+ quenching experiments, the fluorescence of NBD-FP with/without TMD peptide in DMPC:DMPG 15:15 μM vesicles at pH 5.0 or 7.4 was measured until the intensity attained a steady value. The final concentration of each peptide was 0.06 μM. An incremental amount of CoCl2 stock solution (0.1 M) was then injected into the cuvette to give final Co2+ concentration in the range 0.04–2.0 mM. Corrections owing to dilution were made to the observed fluorescence intensities. All parameters were the same as those used for NBD-Rho FRET experiments and the data were analyzed using (1).
Tb3+/DPA leakage experiments
The method is based on the enhancement of the lanthanide metal Tb3+ fluorescence when the aromatic chelator DPA is liganded to the ion. Large unilamellar vesicles (LUVs) of POPC containing Tb3+ were prepared as described previously [19, 31, 32].
To quantitate the extent of leakage observed in the Tb3+/DPA assay, FP or TMD peptide or TMD:FP 1:1 complex were added to a solution containing 40 μM POPC/Tb3+, 50 μM DPA, 100 mM NaCl, 10 mM Tris at pH 7.4. The fluorescence was recorded at ambient temperature with excitation and emission wavelengths of 270 and 490 nm, respectively, and 10 nm bandwidth for both excitation and emission. The percentage leakage of Tb3+ was calculated as follows:
Leakage (%) = [(F t - F 0)/(F max - F 0)] × 100 (3)
where F max is obtained by adding 0.05% (v/v) Triton X-100 and F 0 is equivalent to the values for DMSO controls.
SDS-Polyacrylamide gel electrophoresis (SDS-PAGE)
HA2 FP and TMD peptides were dissolved in HFIP and mixed with lysoPC (1-dodecyl-2-hydroxyphosphatidylcholine) in ethanol as described by Tatulian and Tamm . The organic solvents were removed under a stream of nitrogen followed by high vacuum for 1 h. The dried mixtures were then resuspended in either neutral (43 mM imidazole, 35 mM HEPES, pH 7.3) or acidic buffer (80 mM GABA, 20 mM acetic acid, pH 4.8) and sonicated for 6 min before mixing with the Laemmli buffer (pH 6.8) composed of 62.5 mM Tris-HCl, 25% glceryol, 2% SDS and 0.01% Bromophenol Blue. The concentrations of peptide and lysoPC were around 0.5 and 3 mM, respectively, and the pH of the acidic buffer mixed with Laemmli buffer was raised to about 5.3. For each lane of sample loading, 5 μl of the peptide/lysoPC was mixed with 10 μl Laemmli buffer, except that in lane 3, 5 μl of each of the TMD and FP in lysoPC were mixed before added to 20 μl Laemmli buffer. The molecular weight of each peptide is indicated in parentheses in Figure 2C. Electrophoresis was conducted at 20 mA constant current for 90 min. The image shows the peptide migration in 18% separating gel with 0.1% SDS (pH 8.8). FP exhibits less tendency than TMD to form oligomers in SDS in either neutral or acidic buffer, as shown in lanes 1 and 2. In contrast, TMD formed multiple oligomeric species (lane 4). The association between TMD and FP is not strong enough to counter the dispersing force of SDS detergent and the electric field gradient as seen in lane 3.
Each of the tested peptides was homogenized in a small quantity of HFIP and incubated in pH 5.0 PBS-buffered SUVs to make a final L/P = 50. The sample was subsequently spread on the germanium surface until solvent had evaporated completely. The ATR sample covered with a homemade box was kept in full D2O hydration (D2O/lipid ratio >35 based on the ratio of absorbance peaks of D-O/C-H stretching).
ATR-FTIR spectra were recorded on a BIO-RAD FTS-60A spectrometer equipped with a KBr beamsplitter and a liquid nitrogen-cooled MCT detector. The incoming radiation was polarized with a germanium single diamond polarizer (Harrick, Ossining, NY, USA). The 45° germanium ATR-plate (2 mm × 5 mm × 50 mm) was cleaned using a plasma cleaner (Harrick) before depositing the sample. After 300 scans at a spectral resolution of 2 cm-1, the data were smoothed with triangular apodization and the absorption peaks were analyzed using the Peakfit program to obtain the secondary structure components .
The infrared linear dichroic ratio is defined by R ATR = A ∥/A ⊥ [34, 35], where A ∥ and A ⊥ are the absorbances at parallel and perpendicular polarizations of the incident infrared light, respectively. The tilt angles, relative to the membrane director, of lipid acyl chain (δ), α-helix molecular axis direction (θ) and β-strand axis (Φ) were calculated from equations described in Additional file 1.
attenuated total reflectance
fluorescence resonance energy transfer
- P/L :
peptide to lipid ratio
- SDS PAGE:
sodium dodecyl sulfate polyacrylamide gel electrophoresis
Financial support from Academia Sinica and the National Science Council (NSC 95-2113-M-001-011) of the Republic of China is gratefully acknowledged.
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