Structure and membrane interaction of the internal fusion peptideof avian sarcoma leukosis virus Shu-Fang Cheng, Cheng-Wei Wu, Eric Assen B Kantchev and Ding-Kwo Chang Institute of Chemi
Trang 1Structure and membrane interaction of the internal fusion peptide
of avian sarcoma leukosis virus
Shu-Fang Cheng, Cheng-Wei Wu, Eric Assen B Kantchev and Ding-Kwo Chang
Institute of Chemistry, Academia Sinica, Taipei, Taiwan, Republic of China
The structure and membrane interaction of the internal
fusion peptide (IFP) fragment of the avian sarcoma and
leucosis virus (ASLV) envelope glycoprotein was studied by
an array of biophysical methods The peptide was found to
induce lipid mixing of vesicles more strongly than the fusion
peptide derived from the N-terminal fusion peptide of
influenza virus (HA2-FP) It was observed that the helical
structure was enhanced in association with the model
membranes, particularly in the N-terminal portion of the
peptide According to the infrared study, the peptide inserted
into the membrane in an oblique orientation, but less deeply
than the influenza HA2-FP Analysis of NMR data in
sodium dodecyl sulfate micelle suspension revealed that
Pro13 of the peptide was located near the micelle–water
interface A type II b-turn was deduced from NMR data for the peptide in aqueous medium, demonstrating a conforma-tional flexibility of the IFP in analogy to the N-terminal FP such as that of gp41 A loose and multimodal self-assembly was deduced from the rhodamine fluorescence self-quench-ing experiments for the peptide bound to the membrane bilayer Oligomerization of the peptide and its variants can also be observed in the electrophoretic experiments, sug-gesting a property in common with other N-terminal FP of class I fusion proteins
Keywords: membrane fusion; conformational change; insertion depth; self-assembly; fluorescence self-quenching
Entry of enveloped viruses into the host cells is mediated
by the viral envelope glycoproteins [1], which in most
cases are cleaved by proteolysis to yield the
transmem-brane (TM) [2,3] subunit responsible for memtransmem-brane fusion
and the surface (SU) subunit for receptor binding For a
majority of the class I fusion proteins, a region in the TM
protein crucial for binding to and destabilizing target
membranes, termed fusion peptide (FP), is located at the
N-terminal region, while others have the internal fusion
peptide (IFP) domain [4] Avain sarcoma/leucosis virus
(ASLV) is a prototype retrovirus [5], the envelope
glycoprotein of which uses IFP for fusion to target cells
[6,7] A proline is often found near the centre of many of
the viral IFP sequences [1] Delos et al [8] have shown that the central proline of the FP of ASLV subtype A plays important roles in forming a native envelope protein (EnvA) structure and in membrane fusion It is thought that the envelope protein undergoes conformational change triggered by its binding to the receptor on the target cell surface (e.g Tva for ASLV-A), exposing the hydrophobic FP domain to destabilize the cell membrane preceding the membrane fusion [9] similar to influenza haemagglutinin and HIV-1 gp41 As the majority of studies were performed on the N-terminal FP, it would
be of interest to compare the structure of the internal FP and its interaction with membrane bilayer, including in particular the structural influence of proline Consistent with other class I viral fusion proteins, the IFP of ASLV inserts into the membrane primarily as a helix in contrast
to the IFP of class II fusion protein which uses a cd loop
to insert into the target membrane in the fusion process [10,11] In the following, a variety of physical properties of the putative IFP of ASLV are reported and differences between N-terminal and internal FP are compared The
pH dependence of some of the properties is discussed in regard to the experimental observation that ASLV induced hemifusion, but not complete fusion, at neutral
pH [12]
Experimental procedures All chemicals and solvents were used without further purification N-a-(9-Fluorenylmethoxycarbonyl) (Fmoc)-protected amino acids were products of Anaspec (San Jose, CA, USA) or Bachem (Bubendorf, Switzerland) 1,2-Dimyristoyl-sn-glycero-3-phosphocholine (DMPC) and
Correspondence to D.-K Chang, Institute of Chemistry, Academia
Sinica, Taipei, Taiwan 115, Republic of China.
Fax: + 886 2 27831237, Tel.: + 886 2 27898594,
E-mail: dkc@chem.sinica.edu.tw
Abbreviations: ASLV, avain/sarcoma leucosis virus; ASLV-A, ASLV
subtype A; ATR-FTIR, attenuated total reflectance-FTIR; DG,
dis-tance geometry; DMPC,
1,2-dimyristoyl-sn-glycero-3-phosphocho-line; DMPG, 1,2-dimyristoyl-sn-glycero-3-phosphoglycerol; EnvA,
native envelope protein; FP, fusion peptide; HA2-FP, N-terminal
fusion peptide of influenza virus; IFP, internal fusion peptide; NBD,
7-nitrobenz-2-oxa-1,3-diazole; NBD-PE,
N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)-1,2-dihexadecanyol-sn-glycero-3-phosphoethanolamine;
rhodamine, 5(6)-carboxytetramethylrhodamine; Rh-PE,
Lissa-mineTMrhodamine B
1,2-dihexadecanoyl-sn-glycero-3-phospho-ethanolamine, triethylammonium salt; SA, simulated annealing;
SU, surface; TM, transmembrane.
(Received 26 May 2004, revised 6 October 2004,
accepted 13 October 2004)
Trang 21,2-dimyristoyl-sn-glycero-3-phosphoglycerol (DMPG)
were obtained from Avanti Polar Lipids (Alabaster,
AL, USA) 7-Nitrobenz-2-oxa-1,3-diazole (NBD) and
proteinase K were purchased from Sigma (St Louis, MO,
USA) 5(6)-Carboxytetramethylrhodamine (TAMRA),
N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)-1,2-dihexadecanyol-sn-glycero-3-phosphoethanolamine (NBD-PE) and
LissamineTM rhodamine B
1,2-dihexadecanoyl-sn-glycero-3-phosphoethanolamine, triethylammonium salt (Rho-PE)
were purchased from Molecular Probes, Inc (Eugene,
OR, USA) SDS and d25-SDS were acquired from
Boeh-ringer Mannheim (Mannheim, Germany) and Cambridge
Isotope (Andover, MA, USA), respectively Solutions
con-taining vesicles were prepared by solubilizing the lipids in
chloroform/methanol (4 : 1, v/v) mixture and drying the
sample under nitrogen stream before dissolving in buffer
solution Peptide/SDS mixtures and peptide/phospholipid
mixtures were sonicated for 30–60 min before
measure-ments
The internal fusion peptide Ac-GPTARIFASILAPG
VAAAQALREIERLA-NH2 (IFP–wt), residues 16–43 of
the native envelope protein from the avian sarcoma and
leukosis virus subtype A, and its P13F (IFP–F13), P13V
(IFP–V13) variants were assembled by Fmoc/t-Bu solid
phase peptide synthesis as C-terminal carboxyamides on
Fmoc-Rink amide Resin using a peptide synthesizer
(Pro-tein Technologies, Tucson, AZ, USA, model Rainin PS3)
operated in the manual mode The N-terminal free peptides
were labelled with TAMRA following the standard
Fmoc-amino acid coupling protocol with a coupling time of 10 h
Labelling with NBD was achieved according the procedure
of Rapaport and Shai [13] with modifications as described in
a recent study [14] Cleavage and purification of the peptide
were as described previously [14,15]
The N-terminal peptide corresponding to residues 1–25
of HA2 (strain X31) of influenza virus, HA2[1–25], was also
synthesized [15] for comparison of structure and function
Circular dichroism experiments
CD measurements were carried out on a Jasco 720
spectropolarimeter Each of the peptides tested was
incu-bated with NaCl/Pi or DMPC/DMPG (1 : 1) vesicular
suspension, at pH 5.0 or 7.4 to give a final concentration of
30 lMof peptide in NaCl/Pior of peptide/DMPC/DMPG
(12 lM: 0.8 mM: 0.8 mM) All samples were measured
with a 1.0-mm path length cell, at 37C The spectra were
recorded from 260 to 190 nm at a scanning rate of
50 nmÆmin)1 with a time constant of 2 s, step resolution
of 0.1 nm, and bandwidth of 1 nm The final spectra were
taken from the average of five scans The VARSELEC
program was used for the secondary structure prediction
as described previously [16,17]
Fluorescence spectrometry
All fluorescence experiments were performed on a Hitachi
F-2500 Fluorescence Spectrometer at 37C using a 1 cm2
semimicro quartz cuvette with stirrer The response time
was set at 0.08 s, slit bandwidth for excitation and emission
was 10 nm A scan rate of 300 nmÆmin)1was used for the
wavelength scans
Membrane binding and depth of immersion
of the peptide probed by N-terminally labelled NBD The NBD-labelled peptide was used to monitor the interaction between the peptide and lipid vesicles of DMPC/DMPG (1 : 1, molar ratio) with a suspension containing 0.06 lM and 300 lM of the NBD-labelled peptide and phospholipid, respectively, at pH 5.0 or 7.4 [18] The excitation and emission wavelengths were set at
467 and 530 nm, respectively, for time scan measure-ments Spectra in the 500–650 nm range were collected in the wavelength scan experiments The digestive enzyme, proteinase K (60 lgÆmL)1 in final concentration), was added to vesicles loaded with NBD-conjugated peptide
to investigate the extent of protection from the enzyme action by the membrane on each of the peptides tested
For Co2+ quenching experiments, the NBD-labelled peptide was added to a cuvette containing DMPC/DMPG vesicles at pH 5 or pH 7.4 and measurement was taken until the fluorescence signal attained a steady value The final concentrations of peptide/DMPC/DMPG were 0.06 : 150 :
150 lM An incremental amount of CoCl2 stock solution (0.1M) was then injected into the cuvette to give final concentrations in the range of 0.02–2.0 mM Corrections due
to dilution were made to the observed fluorescence intensi-ties The data were analyzed using the Stern–Volmer equation:
F0=F¼ 1 þ Ksv[Q]
where F0 and F are the intensity of NBD fluorescence before and after adding a given amount of CoCl2 solution, respectively, [Q] is the concentration of the quencher and the slope KSV is the Stern–Volmer constant
Lipid mixing assay by fluorescence resonance energy transfer
Membrane fusion assay used in the study is based on the measurement of FRET from NBD to rhodamine [19] Specifically, two lipid suspensions were prepared, one unlabelled (DMPC/DMPG 250 : 250 lM) and one labelled (DMPC/DMPG/NBD-PE–Rho-PE 250 :
250 : 5 : 5 lM), using NaCl/Pibuffer at neutral or acidic
pH A 9 : 1 molar ratio of unlabelled to labelled liposomes (total volume 1 mL) was used in the assay; hence the final DMPC/DMPG to NBD-PE molar ratio is
1000 after the lipid mixing Various aliquots of 1 mM peptide stock solution dissolved in dimethylsulfoxide (DMSO) were injected into the liposome mixture As a control, 20 lL DMSO was used As the fusion peptide is added to induce lipid mixing, the fluorescent probe is diluted by mixing of the unlabelled and labelled vesicles, resulting in reduced energy transfer efficiency and an increase in the fluorescence intensity of the energy donor, NBD-PE To monitor the NBD probe, the excitation and the emission wavelengths were set at 467 nm and 530 nm, respectively The fluorescence intensity after the addition
of Triton X-100 (0.2% v/v) was referred to as 100%, respectively
Trang 3Self-association tendency of IFP–wt by N-terminally
labelled rhodamine fluorophore
The rhodamine self-quenching experiments were carried out
to examine the propensity of self-association of the peptides
in NaCl/Pi and in the vesicular suspension Briefly, the
rhodamine-labelled peptide (0.1 lM) in aqueous buffer at
pH 5.0 or 7.4 was mixed with DMPC/DMPG (1 : 1, molar
ratio) vesicles (lipid concentration 200 lM) To monitor the
rhodamine probe, the excitation and emission wavelengths
were set at 530 and 578 nm, respectively Digestion of the
labelled peptides by proteinase K (50 lgÆmL)1) leads to
disassembly of the membrane-associated oligomeric fusion
peptides, resulting in dequenching of rhodamine
fluores-cence The 100% reference intensity was taken from the
fluorescence measured in the peptide/lipid dispersion
solu-bilized with 0.2% (v/v) Triton X-100
In the experiments on the composition variation of
rhodamine-labelled peptide, the total (labelled plus
unla-belled) peptide concentration was kept constant at 0.01 lM
while the fraction of labelled peptide, x, was varied from
0.05 to 1 in DMPC/DMPG (150 : 150 lM) vesicular
suspension The normalized emission intensity Ix/x was
plotted against x [20]
It is noted that intra-trimeric interaction is detected
for x values near 1 since nearly all peptide molecules are
labelled and quenching therefore arises predominantly from
the close neighbours within the same trimer In contrast, for
low x values, the probability of finding a pair of labelled
peptides is slim and hence quenching arises mainly from
labelled peptides in nearby trimers
SDS/PAGE experiments to examine the oligomerization
of IFP–wt, –F13 and –V13 in the membranous setting
A PhastSystemTM (Pharmacia Biotech, Sweden)
accom-panied with PhastGel high density and PhastGel SDS
buffer strips was used for SDS/PAGE experiments, which is
particularly suitable for molecules in the molecular mass
range 1000–20000 Peptide samples were added to 6% SDS,
10% glycerol and 10 mMTris-buffered solutions at pH 6.8
and heated at 55C for 10 min IFP analogues and markers
(0.5 mM; Pharmacia Biotech MW marker kit, code no
80-1129-83) were loaded in a PhastGelsample applicator
8/1 (code no 18-1816-01) The running condition and
staining method followed the procedures given in
Phast-System handbook (ref no 80-1312-29 and 80-1312-30,
respectively) Compositions of the buffer system in the gel,
buffer strips and solutions used for development can be
found in the homepage http://www.apbiotech.com
Attenuated total reflectance-FTIR measurements
Polarized ATR-FTIR spectra were recorded on a Boman
DA8.3 spectrometer with a KBr beamsplitter and a liquid
nitrogen-cooled MCT detector according to procedures
described previously [20] Each of the studied peptides
(20 lg) and DMPC/DMPG (1 : 1, molar ratio) were mixed
in chloroform/methanol (1 : 1, v/v) solution and
equili-brated with sodium phosphate buffer at pH 5.0 or 7.4 to
give a final peptide/lipid molar ratio of 1 : 50 The sample
was carefully spread on the germanium surface until solvent
had evaporated The ATR sample covered with a home-made box was kept in full D2O hydration (D2O/lipid ratio
> 35) based on infrared absorbance ratio of D-O/C-H stretch peaks
Three hundred scans were collected at a resolution of
2 cm)1with triangular apodization and incoming radiation was polarized with a germanium single diamond polarizer (Harrick, Ossining, NY, USA) Before depositing sample, the 45 germanium ATR-plate (2 · 5 · 50 mm) was cleaned by a plasma cleaner (Harrick, Ossining, NY, USA) Analysis of ATR-FTIR data was performed in accordance with a previous study [21]
NMR experiments NMR samples were prepared by dissolving the IFP–wt powder at 1 mM concentration in H2O/D2O 9 : 1 (v/v) aqueous buffer or 100 mM d25-SDS micellar solution Dilute HCl or NaOH solution was used to adjust pH to 5.0 One- and two-dimensional 1H NOESY and TOCSY NMR experiments were performed on a Bruker AMX-500 spectrometer at 298 K (in SDS micellar solution) or 278 K (in aqueous solution), as described previously [22] In deuteron/hydrogen (D/H) exchange experiments, the pep-tide incorporated into SDS sample was lyophilized three times with pure H2O D2O/H2O 9 : 1 (v/v) was added immediately before acquiring NMR data at 298 K and
pH 5.0 To measure the effect of Mn2+ ions on the relaxation behaviour of IFP–wt protons, MnCl2dissolved
in H2O was added to IFP–wt micellar solution to give a final molar ratio of 0.696 (Mn2+/IFP–wt) Mn2+is an aqueous ionic probe which is excluded from the apolar core of the micelle Because the relaxation rate enhancement varies inversely with the distance between the proton and the probe, the protons located more deeply in the micellar interior will be less affected by the spin probe The fraction
of attenuated backbone amide proton signal is taken as the fractional intensity difference in the cross-peaks of NH/aH
or side-chain protons of a given residue obtained for the protonated and deuterium-exchanged peptide (for exchange experiments) or before and after introduction of Mn2+to
d25-SDS micellar solution (for relaxation enhancement measurements)
Structure calculations Using distance geometry (DG)/simulated annealing (SA) protocols of BIOSYM programs INSIGHTII, DISCOVER and NMRCHITECT (version 2000.1) from Accelrys Inc (San Diego, CA, USA), 360 constraints were employed in the structural computations (Table 1) The intermolecular NOE restraints were classified semiquantitatively into three categories: strong (less than 2.6 A˚), medium (2.6–3.6 A˚) or weak (3.6–4.6 A˚) A range of 0.6–2.0 A˚ was allowed to vary in the distance constraints In the SA protocol, the temperature was raised to 1000 K in four steps followed by
a molecular dynamics run for 30 ps to allow more conformational space to be explored The system was subsequently annealed to 300 K in 10 steps for a total of
55 ps and minimized by the steepest-descent and conju-gated-gradients methods before final refined structures were obtained
Trang 4CD experiments on IFP–wt and -F13 indicate helix
enhancement of the two peptides upon associating with
lipid bilayer
Figure 1 displays CD data on the two internal FP analogues
in aqueous solution and in DMPC/DMPG (1 : 1) vesicular
dispersions at pH 5.0 and 7.4 at 37C No significant change
in the secondary structure occurs upon acidification from
pH 7.4–5.0 for both analogues in DMPC/DMPG
disper-sion; the only peculiarity is a dramatic increase in helicity
with acidic pH for the F13 analogue in aqueous solution
On the other hand, the helix content is increased when IFP–
wt is transferred from aqueous to vesicular suspension, in
analogy to the N-terminal FP such as that of gp41 [22] and
of influenza HA2 [23], demonstrating the conformational
plasticity of the viral fusion peptides However, the helix
population in the membranous environment is higher for
IFP–wt than for these two N-terminal FPs The high helix
content of F13 variant in aqueous solution at acidic pH may
reflect the critical effect of proline on the helical propensity
of the fusion peptide sequence, but this structural effect
diminished on associating with the membrane As will be
further demonstrated by NMR results, helix is induced in
the N-terminal portion of the peptide in the membranous
environment
Binding of IFP–wt to membrane bilayer is detected
by NBD-labelled peptide
To investigate the nature of membrane interaction of IFP–wt
as manifested in the secondary structure change of the
peptide upon binding to the membrane (Fig 1), we utilized IFP–wt with an N-terminally attached NBD which exhibits greatly increased fluorescence emission in a less polar environment As illustrated in Fig 2A, the fluorescence intensity increased several fold when the labelled peptide was transferred from aqueous buffer to vesicle dispersions, providing direct evidence that the peptide (or at least its N-terminal portion) penetrates into the membrane apolar interior To further investigate the insertion depth, Stern– Volmer constant KSV obtained from NBD quenching by
Co2+was utilized The KSVwas calculated from the linear part of Fig 2B (in the range of [Co2+]¼ 0–0.3 mM) The data reveal that acidification of vesicular dispersion results in deeper immersion of the peptide, at least at the N-terminus,
as reflected by a smaller KSV In the bottom panel of Fig 2B, data from HA2(1–25) were used to compare the insertion depth between the N-terminal and internal fusion peptides The N-terminal region of the N-terminal fusion peptide is seen to penetrate more deeply than that of IFP–wt The result
Table 1 Constraints used for molecular simulation calculations on
IFP–wt in sodium dodecyl sulfate micelle and the deviations from the
average structures.
Constraint type
Total no.
constraints
No.
constraints Constraints
Subtype
Medium
RMSD type
Deviation range No of deviation Distance 0.5–1.0 A˚ 17
> 1.0 A˚ 0 Dihedral > 5 19
> 10 7
> 15 7
> 20 0
a
d(i,j) constraints where j 3 i+5.
Fig 1 Far-UV CD spectra of ASLV IFP–wt and IFP–F13 in aqueous buffer and in DMPC/DMPG vesicle media at pH 5.0 (top panel) and 7.4 (bottom panel) at 37 °C Helix content estimated from the ellipticity value at 222 nm is enhanced considerably as IFP–wt was transferred from aqueous to vesicular solution at both pHs; this is true for IFP–F13 only at pH 7.4 The CD spectra of IFP–V13 are similar to those of IFP– F13 under the same conditions The insets to the panels display the results of the secondary structure analysis with the VARSELEC program.
Trang 5will be discussed in conjunction with NMR data (Fig 7)
obtained in SDS micellar solution Furthermore, greater
insertion depth is observed for both fusion peptides at acidic
pH than at neutral pH This may have implications on the
fusion phenotype of the viral fusion proteins since the fusion
function of both envelope proteins is sensitive to pH [24]
Fluorescence resonance energy transfer data demonstrate the lipid mixing activity of IFP–wt
To investigate the fusogenicity of the peptide, NBD- and Rho-labelled PE were used as the donor and acceptor, respectively, of fluorescence energy transfer Dilution of the fluorescent-labelled PE loaded into vesicles by the unloaded vesicles via membrane fusion induced by the fusion peptide results in a reduction in the fluorescence energy transfer efficiency, hence dequenching of the donor fluorescence As illustrated in Fig 3, lipid mixing as defined in Experimental procedures is plotted against peptide to lipid ratio indicates that the peptide is capable of promoting lipid mixing of vesicles consisting of DMPC and DMPG Additionally, a peptide derived from randomized sequence of IFP–wt (see Supplementary data) exhibited insignificant lipid mixing activity, demonstrating the specificity of the activity of the IFP–wt sequence Inspection of Fig 3 revealed that lipid mixing activity is higher for IFP–wt than the N-terminal fusion peptide, HA2[1–25], of influenza virus at the same peptide-to-lipid ratio The result is analogous to the data on the Sendai virus observed by Peisajovich et al [25] showing that the IFP has higher fusion activity than the N-terminal FP
Self-quenching of rhodamine fluorescence indicates self-assembly of IFP–wt in the lipid bilayer
To further examine the organization of the fusion peptide in the membrane, the peptide was labelled with rhodamine [26] Self-association of the molecules is monitored by the
Fig 2 Membrane binding and insertion depth of IFP–wt probed by
NBD fluorescence (A) Lipid binding of NBD-IFP–wt at pH 7.4 and
37 C Increased intensity and the blue-shift of fluorescence of NBD
attached to the N-terminus of the peptide indicate embedding of the
peptide in the apolar milieu of membrane bilayer As a control,
pro-teinase K digestion of the peptide disrupts membrane binding releasing
bound NBD and thus reduces fluorescence of the fluorophore (B)
Stern–Volmer plot of cobalt quenching of NBD-IFP–wt to probe the
immersion depth in DMPC/DMPG vesicular suspension at pH 5.0
and 7.4 K SV values of HA2(1–25), the fusion peptide of influenza virus
are compared to IFP–wt The calculated K SV values (based on the data
in the range of [Co 2+ ] ¼ 0–0.3 m M ) were shown in the inset For both
fusion peptides, K SV is smaller at lower pH, indicating deeper
penet-ration than at neutral pH At the same pH, larger K SV for IFP–wt
shows shallower immersion of the peptide in the vesicle than the
N-terminal FP.
Fig 3 Lipid mixing induced by IFP–wt as probed by FRET at 37 °C NBD- and Rho-labelled PE were incorporated in the vesicles to which were added fusion peptide and unlabelled lipid dispersion Mixing rates are plotted against peptide-to-lipid ratios for IFP–wt and the N-terminal FP of influenza virus Fusion activity exhibits strong
pH dependence for the IFP–wt Lipid mixing rate is larger for IFP–wt than the influenza fusion peptide at a given P/L-value Dequenching
of the donor NBD by dilution of the acceptor Rho resulting from peptide-mediated membrane fusion is normalized with respect
to the intensity obtained from lysis of vesicles with 0.2% Triton X-100.
Trang 6self-quenching of rhodamine fluorescence The result shown
in Fig 4A for IFP–wt in DMPC/DMPG vesicular
suspen-sion indicates a moderate (approximately 40%)
dequench-ing after addition of the detergent (Triton X-100) to the
suspension, suggestive of a loose association for the peptide
in the membrane bilayer
Self-assembly can also be analyzed by compositional variation of rhodamine-labelled peptide, keeping the total concentration of labelled and unlabelled peptides constant
In the experiments leading to Figs 4B, 0.01 lM of total peptide was incorporated in DMPC/DMPG (150 : 150 lM) vesicle dispersion for IFP–wt, with composition of the labelled peptide (x) varying from x¼ 1–0.05 All three peptides exhibit characteristic of multimeric species in the lipid bilayer, as displayed by self-quenching of rhodamine at
x¼ 1 compared to intensity at x ¼ 0.05 (for example, quenching efficiency in excess of 3 for IFP–wt) and the shape of Ix/x vs x plot Specifically, the initial slow rise of the latter plot (from x¼ 1) reflects that the self-association
of the peptide is not tight (on the scale of self-quenching distance of rhodamine, 15 A˚ [17]) The deviation of the observed profiles from calculated ones based on homogen-eous clustering of various multimers (N¼ 1, 2, 4 and 8) indicates more than one mode of association of the FP molecules, involving more tightly packed oligomeric (such
as trimer as in other class I fusion proteins) subunits interacting loosely with neighbouring oligomers Thus, the sharp increase in Ix/x near the x¼ 0 region shows dequenching of the probe and hence a tendency toward random distribution of the peptide in the membrane in the long distance range (> 30 A˚), indicating no large scale aggregation occurs for the peptide
SDS/PAGE experiments suggest propensity of self-association of IFP–wt in the membrane-mimic environment
Self-assembly of IFP–wt in the membranous environment can also be discerned by SDS/PAGE data shown in Fig 5, which also displays the results for IFP–F13 and IFP–V13 All peptide analogues exhibit diffuse bands and migrate roughly as dimeric species, with F13 and V13 variants forming oliogomers of slightly higher mass The data are in qualitative agreement with the rhodamine self-quenching result for IFP–wt (Fig 4) All peptides exhibit little tendency of forming a large and tight molecular cluster
As SDS micelle is considered to be strongly disruptive on
Fig 4 Self-assembly of the internal fusion peptide analogues in
associ-ation with DMPC/DMPG vesicles at pH 7.4 and 37 °C (A) Relative
intensity of Rho-labelled IFP–wt, –F13 and –V13 in aqueous buffer
(unfilled bar), DMPC/DMPG vesicle (grey bar) and vesicle treated
with proteinase K (black bar) The results indicate that IFP–wt has
lower propensity of forming oligomer in the vesicular medium than the
other two variants and is probably monomeric in aqueous solution as
Rho self-quenching is less than in the presence of vesicles for the
labelled peptide (B) Normalized Rho emission intensity as a function
of the fraction of labelled peptide as a probe for self-aggregation As
indicated in the plot, monomeric and dimeric species for the peptide
are represented by the hypothetical horizontal and diagonal lines,
respectively The nearly unchanged I x /x in the high x region reflects a
tight shorter range, probably intratrimeric, packing but the sharp rise
in the low x region indicates a loose association for longer range
inter-trimeric interaction This interpretation is based on the fact that, at
low x limit, the probability of finding a labelled FP within a trimer for
a given rhodamine probe is low thus the result emphasizes inter-trimer
interaction; the reverse is true in the x ¼ 1 limit Moreover, the
devi-ation of the experimental curves from those calculated by assuming a
single species of association of N monomers indicates a multi-mode
association for the peptide analogues in the membrane bilayer The
data therefore imply a heterogeneous distribution of FP molecules,
which can be interpreted by a more close-packed trimers interacting
loosely with adjacent trimers.
Fig 5 SDS/PAGE measurements on the molecular association for the three IFP analogues In accord with the data of Fig 4A, IFP–wt has lower propensity of forming high order oligomer than the other two analogues.
Trang 7the non–covalent interaction, the data of Fig 4 provide indirect evidence for propensity of IFP–wt and analogues for self-assembly in the membranous medium
Helical structure and insertion angle
of the membrane-associated fusion peptide
as measured by FT-IR spectroscopy The secondary structure of peptides can also be quantitated
by infrared spectroscopy As shown in Fig 6, the helical content calculated from the band at 1655 cm)1 after deconvolution is 60% (Table 2), in agreement with that observed in the CD data of the wild-type peptide in the vesicular suspension at pH 7.4 (cf Figure 1) The helix population in IFP–wt is higher, while the b-sheet content is lower, than that in the fusion peptide of influenza virus [20] Slightly higher helix content was found for IFP–F13 (64%, Table 2), and IFP–V13 (66%, data not shown), suggesting helix structure is not a sufficient determinant for the fusion activity of the internal fusion peptide of ASLV Secondary structure composition of the peptide varied little, if any, with pH between 5.0 and 7.4 (C.W Wu and D.K Chang, unpublished observation)
The insertion angle of the peptide helix deduced from the polarized ATR FT-IR result (Table 2) is 53 with respect
to the membrane normal Compared to N-terminal fusion peptides such as that of influenza virus [20], the result indicates that IFP–wt associates with the bilayer in a more shallow fashion Still shallower insertion was obtained for the IFP–F13 variant There is an insignificant change in insertion angle as pH varies from 5.0 to 7.4
Secondary structure change and insertion depth for membrane-bound fusion peptide can be determined
on the residue level by NMR measurements The secondary structure and membrane insertion of the fusion peptide can be examined at the atomic level by NMR spectroscopy using a micelle as a model Figure 7A summarizes the NOE interactions from proton pairs with distance¼ 5 A˚ in SDS micellar dispersion and in aqueous buffer solution at pH 5.0 The daN(i,i+3) and daN(i,i+4) interactions, characteristic of helix structure, can be found in the residues 1–10 and residues downstream of Pro13 in SDS micelles In accord with NOE data, helical segments, which
Fig 6 ATR-FTIR results on IFP–wt in DMPC/DMPG vesicles at
pH 7.4 The top panel displays data from polarized FT-IR
experi-ments; the ratio of the parallel to perpendicular components relates to
the angle of insertion of the FP molecules into the bilayer || and ^
represent parallel and perpendicular polarized light, respectively The
bottom panel displays the result of deconvolution of the IR absorption
spectrum of the peptide for analysis of the secondary structure The
dominant peak at 1655 cm)1is attributed to the helical structure The
dotted and solid traces are experimental and deconvoluted data,
respectively.
Table 2 Secondary structure and helix orientation of IFP–wt and -F13 in DMPC/DMPG vesicular dispersion as deduced from ATR-FTIR data a The peptide-to-lipid molar ratio was 1 : 50.bH values are angles between the helix axis and the bilayer normal.
Secondary structure percentage
Helix axis orientation
Order parameter Samide I¢ 0.032 ± 0.02 )0.19 ± 0.03 0.095 ± 0.04 )0.14 ± 0.06
Trang 8are characterized by consecutive residues with spin–spin coupling constant3JaN¼ 5 Hz, can be identified for the residues 3–10 and 14–28 as shown in Fig 7B Also plotted
in Fig 7B are 3JaN data in aqueous buffer, indicating residues 16–21 are more helical while residues 6–10 have more b-strand character Comparing3JaNvalues in aqueous buffer and in micellar suspension, it is clear that helix is induced in the N-terminal half for the peptide in association with SDS micelle Thus helix enhancement of the peptide on binding to the membrane shown in Fig 1 is corroborated
by the data of Fig 7
The backbone amide deuterium/proton (D/H) exchange can be used to probe the immersion of the peptide in SDS micelle with the notion that slower exchange correlates with deeper penetration into the micelle for the residue under consideration Another method to gauge the insertion depth
is a reduction of resonance intensity resulting from relax-ation enhancement by the aqueous spin probe, manganese, the extent of which is highly dependent on the Mn2+– proton distance [27] The results of these two experiments as summarized in Fig 7C are consistent in that the region encompassing Pro13 is near the micellar headgroup region and the residues 16–21 penetrate more deeply into the micellar interior than other regions of the peptide Accord-ing to Fig 7C, the backbone of Arg22 resides near the apolar–headgroup interface of the micelle; its side chain is likely extended to the headgroup region of the micelle resulting in neutralization of the positive charge by the sulfate group
Fig 7 Secondary structure and topology of IFP–wt in SDS micelles
determined by NMR spectroscopy (A) NOE interactions of IFP–wt in
aqueous buffer (top) and in association with SDS micelles (bottom).
(Upper) Folded structure can be discerned in the region 10–18 and 20–
26 (Lower) Helical segments can be observed in 1–11 and 14–28 by
contiguous (i,i +3) interactions The absence of (i,i +3) cross-peaks
between Leu11 and Gly14 is consistent with a distorted helix for the
region around Pro13 (B)1H spin–spin coupling constants (3J aN ) for
the residues of IFP–wt measured in aqueous solution and SDS micellar
suspension Residues of the helical and b-sheet structure are
charac-terized by values smaller than 5 Hz and greater than 8 Hz,
respect-ively The transformation into helix of the N-terminal regions is
obvious as IFP–wt is transferred from aqueous to micellar medium.
(C) Attenuation of the intensity of NH-aH cross-peaks in D/H
exchange and Mn 2+ relaxation enhancement experiments The
standard error in computing signal attenuation is 5% More exposed
backbone amide proton results in faster exchange between deuterium
and proton and hence smaller cross-peak retention The relaxation
enhancement of the backbone protons is inversely proportional to the
sixth power of Mn2+-proton distance; larger signal retention therefore
represents greater depth of insertion of the amino acid residue into the
micelle Taken together, the two sets of data indicate that the stretch
around Pro13 is closer to the surface and the C-terminal half penetrates
more deeply than the N-terminal half of IFP–wt.
Fig 8 NH-aH and NH-side chain proton region of1H NOESY spec-trum of IFP–wt in the aqueous medium Interactions between the resi-dues around Pro13 used to determine the type II b-turn are indicated Particularly noteworthy are the cross-peaks attributed to bH of Ala12 and NH of Val15, and aH of Pro13 and NH of Val15.
Trang 9Conformational alteration of IFP–wt occurs when the
peptide is transferred from aqueous solution to SDS
micellar dispersion, particularly in the region near Pro13
NMR data of IFP–wt suggest an overall lower helicity in
aqueous solution compared to that in SDS micellar
solution Because of the helix-disruptive property of proline,
we further examined the structure of the region adjacent to
Pro13 of IFP–wt Several lines of evidence from NMR data
shown in Fig 8 suggest that Pro13-Gly14 essentially form a
type II b-turn in aqueous solution, while a slightly kinked
helix is deduced in the presence of micelles First, in aqueous
medium coupling constant3JaNfor Gly14 is 6 Hz, closed
to 5 Hz in a type II b-turn Second, the result that
daN(Pro13,Gly14) is stronger than daN(Gly14,Gly14)
indi-cates that a type II instead of type 1 turn is adopted for the
Ala12-Pro13-Gly14-Val15 stretch Third, the relationship of
interproton distance also provides evidence of the claimed
turn structure Thus dNN(Gly14,Val15) is short for the
strong intensity of the cross-peak between the amide
protons of the two residues; daN(Gly14,Val15) is shorter
than intraresidue daN(Val15,Val15); a prominent dbN
(Ala12,Val15) peak is observed but not other dbN(i,i +3)
for i¼ Ser9, Ile10 and Leu11, suggesting a b-turn exists in
the segment Ala12-Pro13-Gly14-Val15 A turn-to-helix
conformational change is also manifested in the upfield
shift of Val15 CaH from 4.05 to 3.85 p.p.m as SDS micelles
are added to the medium although the helix is distorted near
Pro13 in the presence of the micelle Another result in
support of the contention that Val15 is the fourth residue of
a turn is afforded by the small temperature coefficient of
NH of Val15, namely 2.5· 10)3p.p.m.ÆC)1(compared to
8.5· 10)3p.p.m.ÆC)1for Gly14 NH) in aqueous solution,
suggesting that its NH participates in hydrogen bonding The latter notion is also corroborated by a distinct
daN(Pro13,Val15) cross-peak
A kink at Pro13 is visualized by the structural calculation based on the proton NOE and coupling constant constraints
To obtain three-dimensional structure, we employed INSIGHTII/DISCOVER and NMRCHITECT using constraints derived from NOE and 3JaN Figure 9 illustrates the superposition of 20 structures (PDB ID: 1XNL) Detailed structural statistics are tabulated in Table 1 Note that the helices have a kink of 20 at Pro13, consistent with the effect of the proline on the helix summarized by Barlow and Thornton [28]
Discussion
We have studied the structure of the IFP of ASLV-A and its membrane interactions to examine the difference in the biophysical property between the N-terminal and internal FPs, in the hope of improving our understanding of the role
of fusion peptides in the fusion event
Structure and membrane interaction of IFP–wt Binding of IFP–wt to lipid vesicles as demonstrated in Fig 2 supports the view that the IFP of ASLV is either responsible for or contributes to the binding of the virus to liposomes after exposure of the fusion peptide in response to the conformational change triggered by interaction with the specific receptor [8]
As is the case for many IFPs, a proline is located near the centre of the FP sequence Both CD and NMR data indicate a higher helix content than the N-terminal FP (e.g influenza HA2 and HIV-1 gp41) in the membrane-mimic environment Figures 1, 7 and 8 also demonstrate b–a transformation when the peptide is transferred from aque-ous to micellar medium; more specifically, NMR results indicate that the helical structure is induced mainly in the region N-terminal to proline and there is a slight kink in the helix at Pro13 As shown in Fig 7C, the region surrounding Pro13 resides closest to the micellar surface A kinked helix structure has also been deduced from an NMR study on the fusion peptide of HA2 in dodecylphosphocholine micellar solution [29], indicating that it is therefore probably a common feature for the class I viral fusion peptides embedded in the membrane and is involved in destabilizing the membrane during the fusion process The notion is supported by a mutational study on ASLV fusion protein in which the viral infectivity was found largely unaffected by the substitution of Pro13 with a residue thought to retain the bending or flexibility of the central region of IFP [30] Except for the moderate distortion on the helical structure, Pro13 does not significantly reduce helicity of the wt-peptide
in lipid as observed from the comparison of CD data (Fig 1) between the peptide and its F13 variant
The conformational switch for the stretch near Pro13 from a type II b-turn to a kinked helix as the fusion peptide
is transferred from aqueous to SDS micellar medium underscores our previous contention regarding the
Fig 9 Superposition of 20 structures of IFP–wt calculated with the
constraints derived from NMR data performed in SDS micelles A kink
caused by Pro13 shows that the amino acid may have a structural effect
on helix in the membrane-mimic medium similar to that in aqueous
solution.
Trang 10N-terminal fusion peptide that the structural plasticity is
germane to its fusogenicity and a proper balance between
helix and b-sheet structure is important for fusion activity
It is thus likely that both internal and N-terminal fusion
peptides destabilize the target membrane in a similar
fashion, notwithstanding a shallower penetration of the
IFP into the membrane
It is noteworthy that substitution of Pro13 by a residue
with intermediate hydrophobicity (Thr for instance) resulted
in an MLV-pseudotyped virus with higher infectivity [8]; the
activity, moreover, did not correlate with the predicted
secondary structure propensity of the substituted amino
acid The result is in line with our finding that Pro13 is
located in the interfacial region having transitional polarity
Introduction of a residue with extreme polarity may perturb
the topology of the fusion peptide and its interaction with
the membrane bilayer It is of interest to note the phenotype
of HIV-2 gp41 mutants [31]: the amino acid substitution of
the fusion peptide sequence that increases hydrophobicity of
the region would enhance syncytium formation, while
mutation that increases charge or polarity of the fusion
peptide domain reduces syncytium-inducing capacity of the
virus Coupled with our previous finding of a deeper
insertion of the fusion peptide of HIV-1 gp41 into model
membrane [22], the result of Steffy et al [31] corroborates
the assertion that Pro13 of IFP–wt resides near the
interfacial region of the bilayer
The orientation and insertion depth were probed by
FTIR and NMR techniques The angle between the helix
axis and bilayer surface deduced from FTIR data is
37 for IFP–wt ( 27 for IFP–F13) but 50 for
HA2-FP [20] Hence the IFP–wt inserts into bilayer less
steeply than the N-terminal fusion peptide of influenza
virus The insertion depth obtained from NMR data
(D/H exchange and Mn2+ probe) in the SDS micelle
suspension reveals that the N-terminal portion of the
N-terminal fusion peptide [15] is embedded more deeply
than the C-terminal segment whereas the reverse is true
for IFP–wt Moreover, the estimated angle of insertion
from the relaxation enhancement measurements is flatter
and closer to the micelle surface for IFP–wt This
suggests that IFP has a shallower insertion into the
membrane It also follows that penetration into both
leaflets of the membrane bilayer is not required for the
IFP to exert its function The result is reasonable as the
charged and polar residues flanking the IFP sequence
would prevent the FP from immersing too deeply into
the apolar core of the membrane Our data are also
compatible with the observation that truncation of the
membrane-spanning region of HA2 to the extent that the
resulting segment cannot cross the bilayer led to
hemi-fusion but not complete hemi-fusion [32] and with the report
that lipid-anchored influenza haemagglutinin promotes
hemifusion [33]; both imply that the complete fusion
cannot be supported by the fusion peptide domain alone
and the transmembrane segment is indispensable The
deeper penetration at pH 5.0 than at pH 7.4 for both
HA2 and ASLV fusion peptides as revealed by Fig 2B
may be relevant in the pH dependence of fusion activity
for both viruses; it may be that further perturbation of
membrane structure with deeper insertion would promote
progress to later stages of fusion reaction, for example
deeper penetration into membrane at acidic pH may facilitate progress from hemifusion achieved by ASLV at neutral pH to pore formation [34] Comparison of the orientation of IFP–wt and IFP–F13 in association with phospholipid bilayer (Table 2) also suggests that a small angle of insertion into the membrane may exist for the internal fusion peptide [35] to destabilize the membrane
An oblique insertion of fusion peptide of SIV gp32 and influenza HA2 has been deduced by Martin et al [36] and by Lu¨nerberg et al [37] from FT-IR measurements
Lipid mixing activity of IFP–wt and its relationship
to the secondary structure Lipid mixing measurements on the internal and N-terminal fusion peptides indicate that the IFP has higher fusion activity than N-terminal fusion peptide of influenza virus (Fig 3) Both types of fusion peptides insert into the membrane bilayer obliquely and possess substantial regular structures (a-helix and b-sheet) in the membranous medium,
in support of the idea that balanced helix and b-sheet structures are necessary for fusion Indeed, higher helix content alone does not correlate with fusogenicity as can be seen from comparison of helical structure between IFP–wt and IFP–F13
Organization of IFP–wt in model membranes
An apparently dimeric band can be detected in the SDS/ PAGE measurement for IFP–wt A species of slightly higher apparent molecular mass is observed for each of the less fusogenic analogues IFP–F13 and IFP–V13, suggesting that oligomerization of fusion peptide molecules in the membrane is not sufficient for their fusion activity This is in contrast with the oligomerization propensity observed for HA2-FP and its mutants [17] Both the simple rhodamine self-quenching and the rhodamine-labelled peptide compo-sition variation experiments (Fig 4B) clearly demonstrate self-assembly for the membrane-bound peptides; the latter further provided evidence of multimodal self-assembly of the peptide, as deduced, in particular, from a steeper rise in
Ix/x in the region near x¼ 0 The oligomeric structure for the ectodomain of the envelope glycoprotein of ASLV has also been inferred from sucrose gradient sedimentation experiments [38] The results of Fig 4 lead to the view that loose association of the IFP molecules in the membranous environment is relevant to the lipid mixing activity In analogy to the proposed multi-modal association, the clustering of trimeric envelope proteins on the surface of HIV virion has been elegantly shown by Zhu et al [39] in their electron microscopic study Our data suggest that IFP contributes to the self-assembly of the envelope glycoprotein
of ASLV bound to membrane
Effects of pH on the secondary structure and membrane interaction of IFP–wt
Based on studies on the molecular clustering of EnvA fusion protein of ASLV in response to the receptor binding and low pH, Matsuyama et al [24] proposed a fusion mecha-nism for the virus Specifically, low pH promotes proceeding
of fusion from outer monolayer mixing to complete fusion