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Structural Influences: Cholesterol, Drug, and Proton
Binding to Full-Length Influenza A M2 Protein
E. Vindana Ekanayake,1,2 Riqiang Fu,2 and Timothy A. Cross1,2,3,*
1
Department of Chemistry and Biochemistry, 2National High Magnetic Field Lab, and 3Institute of Molecular Biophysics, Florida State
University, Tallahassee, Florida
ABSTRACT The structure and functions of the M2 protein from Influenza A are sensitive to pH, cholesterol, and the antiinfluenza drug Amantadine. This is a tetrameric membrane protein of 97 amino-acid residues that has multiple functions, among
them as a proton-selective channel and facilitator of viral budding, replacing the need for the ESCRT proteins that other viruses
utilize. Here, various amino-acid-specific-labeled samples of the full-length protein were prepared and mixed, so that only interresidue 13C-13C cross peaks between two differently labeled proteins representing interhelical interactions are observed. This
channel is activated at slightly acidic pH values in the endosome when the His37 residues in the middle of the transmembrane
domain take on a þ2 or þ3 charged state. Changes observed here in interhelical distances in the N-terminus can be accounted
for by modest structural changes, and no significant changes in structure were detected in the C-terminal portion of the channel
upon activation of the channel. Amantadine, which blocks proton conductance by binding in the aqueous pore near the N-terminus, however, significantly modifies the tetrameric structure on the opposite side of the membrane. The interactions between
the juxtamembrane amphipathic helix of one monomer and its neighboring monomer observed in the absence of drug are
disrupted in its presence. However, the addition of cholesterol prevents this structural disruption. In fact, strong interactions
are observed between cholesterol and residues in the amphipathic helix, accounting for cholesterol binding adjacent to a native
palmitoylation site and near to an interhelix crevice that is typical of cholesterol binding sites. The resultant stabilization of the
amphipathic helix deep in the bilayer interface facilitates the bilayer curvature that is essential for viral budding.
INTRODUCTION
Since the first membrane protein and peptide structures
obtained in the presence of lipids (1,2), the influence of
the membrane environment on protein structure has been
the subject of considerable interest (3,4). Well before membrane protein structures were characterized, Anfinsen (5)
made it clear that protein structure was the result of the totality of interactions including those within the protein and
between the protein and its environment. Structures of M2
protein constructs from Influenza A that have been characterized in detergent crystal lattices, detergent micelles, proteoliposomes composed of liquid crystalline lipid bilayers,
and raftlike lipid compositions have extended our understanding of the environmental influences on membrane
protein structure (6–14). Here, we present structural restraints obtained from the full-length M2 (M2FL) proton
channel in lipid bilayers describing the structural influence
of cholesterol, protons, and drug binding on the helices of
Submitted October 5, 2015, and accepted for publication November 25,
2015.
*Correspondence: [email protected]
Editor: Francesca Marassi.
http://dx.doi.org/10.1016/j.bpj.2015.11.3529
the M2 conductance domain (M2CD) using solid-state
nuclear magnetic resonance (NMR) spectroscopy.
While details of the M2 structure are still debated, especially those associated with the conductance mechanism
(15–18), the oligomeric structure is well documented as
a left-handed tetramer, with some dimer-of-dimer character from four monomers, each with a single transmembrane (TM) helix followed by an amphipathic helix in a
juxtamembrane position in the viral interior. The structure
of the N- and C-termini of the protein are largely unknown, except for strong evidence for b-sheet content in
the first 20 residues (19) and recent evidence that a few
residues, and maybe many more in the C-terminus, are
disordered (20).
Solid-state NMR (ssNMR) is the primary technique for
characterizing membrane protein structure in nativelike
bilayer environments at atomic resolution. Recent ssNMR
structures include the structure of phospholamban (21)
and its phosphorylated pentameric form (22); a mercury
transporter, MerF (23); a chemokine receptor, CXCR1
(24); the Mycobacterium cell division protein, CrgA (25);
sensory rhodopsin (26); and the trimeric autotransporter,
Ó 2016 Biophysical Society
Biophysical Journal 110, 1391–1399, March 29, 2016 1391
Ekanayake et al.
YadA (27). Many more publications provide detailed structural results on other membrane proteins. Such studies are
still compromised by the simplified models of the very complex native membrane environment. In important efforts to
address this challenge with the M2 protein, Hu et al. (28)
and Cady et al. (29) have modeled the viral lipid environment using sphingomyelin, DPPC, DPPE, and cholesterol
in a molar ratio of 28:21:21:30 (known as VM) or sphingomyelin, POPC, POPE, and cholesterol in a molar ratio
25.6:25.6:25.6:23 (known as VMþ). However, M2CD
does not bind amantadine (AMT, an antiinfluenza drug) in
either of these environments, suggesting that these environments do not represent nativelike M2 environments (29).
From functional assays conducted by Schroeder et al.
(30), AMT inhibits proton flux through M2FL in liquid crystalline lipid bilayers containing 30% cholesterol, so it does
not appear to be the high concentration of cholesterol that
is responsible for the lack of AMT binding in Hong’s studies
(30). Recently, it has become clear that M2 prefers an
environment on the edge of the raftlike domains where
the pyramidal-shaped M2CD conformation induced by its
amphipathic helix interacting with the lipid bilayer facilitates viral budding (30–32). Indeed, a major strength of
the solid-state NMR technology for membrane protein
structure is the flexibility for preparing samples in a wide
variety of pH and ionic strength conditions and lipid
compositions.
Potentially facilitating the location of the M2 protein on
the periphery of raftlike domains are the relatively short
and significantly tilted TM helices that will be better accommodated in the lipid bilayers surrounding the raftlike domains (30). While it has been suggested that M2 binds
cholesterol, many of these claims are based on cholesterol-recognition amino acid-consensus sequence (CRAC)
motifs occurring in the amphipathic helix of numerous influenza strains (33). However, it is unlikely that these sequences are predictive of cholesterol binding, because
these motifs were originally characterized for TM helices.
Furthermore, in the influenza A/Udorn 307/72 H3N2 strain
(the M2 sequence studied here), the amino-acid sequence
does not meet all of the requirements for a CRAC domain.
However, the M2 amphipathic helix has a Cys50 residue
(mutated in our constructs to Ser) that is natively palmitoylated, a feature that is often associated with cholesterol binding (34).
In this article, we demonstrate the application of mixed/
asymmetrically isotopic labeled samples (18,35), in which
two different 13C labeled preparations of M2FL are mixed
in a 1:1 molar ratio to selectively observe cross peaks
between monomers in this tetrameric structure. For this,
13
C-13C dipolar-assisted rotational resonance (DARR)
experiments (36) are performed to observe local interactions between helices. Consequently, no such cross peaks
will arise from within a monomer, and the cross peaks
will only arise when the different labeled monomers are
1392 Biophysical Journal 110, 1391–1399, March 29, 2016
adjacent to each other in the tetrameric structure. The
1:30 molar ratio of protein monomer/lipid represents a
1:120 tetramer/lipid, which is more than adequate to suppress any tetramer-to-tetramer cross peaks, because there
is no propensity for the tetramers to form a higher-order
oligomeric structure. However, the dilute protein and
dilute pairs of labels leads to limited sensitivity for these
experiments.
MATERIALS AND METHODS
Isotopically labeled M2FL protein was expressed with N-terminal histidine-6 tag from the BL21 RP codon plus Escherichia coli. Initial cultures
were grown in rich media. Two liters of cells were pelleted and washed
with 50 mL of M9 minimal media before growing 1 L of M9 culture supplemented with a 100 mg of an isotopically labeled amino acid (Cambridge
Isotope Labs, Tewksbury, MA; Sigma-Aldrich, Miamisburg, OH). Purification was carried out using Empigen-BB solubilized M2FL after cell disruption with a French press. After high-g centrifugation the soluble fraction
was loaded onto a nickel affinity column and purified M2FL was eluted
from the column at pH 8.0 with 0.5% wt/vol octylglucoside detergent in
Tris buffer. This purified protein was then treated with 5 mM dithiothreitol
for obtaining the protein in monomeric form. Additional details are provided in the Supporting Material.
A 4:1 molar ratio of DMPC and DMPG lipids was used for reconstitution
of the protein in lipid bilayers, and when cholesterol was added a molar ratio of 1:4 (cholesterol/other lipids or ~20% cholesterol lipid fraction) was
used. For reconstitution of M2FL into these lipid environments, the lipid
mixture was made 3% (wt/vol) with the octylglucoside followed by the
addition of a 1:1 molar ratio of two different amino acid-labeled M2FL
preparations to yield a protein/lipid of 1:30. The detergent was removed
by dialysis and confirmed by HPLC using evaporative light scattering
detection (37,38). The final buffer was either 20 mM HEPES buffer for
pH 7.5 or 20 mM citrate buffer for pH 5.0. For AMT-binding experiments,
the drug was added after the removal of detergents to the resulting proteoliposome suspension at a 1:2 molar ratio of channel/drug. Labeled cholesterol was obtained from Sigma-Aldrich (Miamisburg, OH).
Proteoliposomes containing M2 were packed into 3.2 mm-thin walled
magic-angle spinning rotors. Spectra were acquired on a Bruker Avance
600.1 MHz (14.1 T) NMR spectrometer (Bruker, Billerica, MA) at 262 K
using an NHMFL Low-E triple resonance probe for minimal sample heating and optimized sensitivity (39,40). The observation of intermonomer
structural restraints were obtained with DARR experiments (36) using mixing times of 300 or 500 ms. Different spinning rates (9–12.2 kHz) were used
to avoid spinning side-band overlap with the potential cross peaks. All 13C
chemical shifts were referenced externally, using 13C0 labeled glycine sample, by setting the carbonyl carbon resonance to 178.4 ppm relative to TMS.
RESULTS AND DISCUSSION
The labeling strategy was based on the M2CD structure
(PDB: 2L0J (10)) and the amino-acid sequence from the
influenza A/Udorn 307/72 H3N2 strain of M2 (Fig. 1 A).
Five different amino acid-specific 13C-labeled protein preparations were expressed and purified using uniformly 13C
labeled Ala, Ile, Leu, Phe, and Val. The distributions of
the labels are shown in Figs. 1 A and in S1. The two-dimensional magic-angle spinning ssNMR spectra are shown
for each of these single amino acid-labeled samples illustrating that there is no observable scrambling of the labels.
Four different mixed labeled samples were prepared by
Structural Influences to M2FL Protein
FIGURE 1 (A) The amino-acid sequence of M2
protein highlighting the 13C amino-acid labels of
the various samples used here. Note that only one
amino-acid type is labeled in each preparation. The
N-terminus (residues 1–22) is extramembranous
and has significant b-sheet structure (19). The TM
helix is residues 23–46. The juxtamembrane amphipathic helix is residues 47–62 and the C-terminus, for
which there is little information about the structure,
is residues 63–97. (B) The PDB: 2L0J structure of
the M2CD (residues 22–62) in the lipid environment
in which it was characterized (10). One of four monomers is colored yellow. For the nonprotein atoms, the
carbons are green and oxygens are red.
combining pairs of amino acid-labeled samples in a 50:50
mixture: Ala-Ile, Ile-Leu, Val-Leu, and Phe-Leu. The first
three samples were prepared to search for unambiguous
interhelical distance restraints between amino acids of
different types in the TM helices and the last sample was
prepared for a similar search between the amphipathic helices and the adjacent monomer.
In considering the cross-peak possibilities, we and others
have found that the TM domain resonances are stronger
than resonances from the terminal regions of the protein.
Consequently, in general it can be anticipated that our
observed cross peaks would be dominated by magnetization
transfer within the conductance domain. In addition, the
only ambiguities for Ala29 and Ala30 residues in the TM helix are two Ala residues in the C-terminus where there is a
single Ile residue (Ile94). This Ile residue is directly involved
in the virus’s mechanism to evade autophagy by binding
the essential autophagy protein, LC3, to the F91xxI94
sequence—an interaction recognized as monomer-monomer protein interaction (41). Consequently, it is very unlikely that this site would be in close proximity to an
adjacent monomer for an Ala-Ile interaction, and therefore
any observed interactions between Ala and Ile residues
are likely to be associated with packing of the TM helices.
The Ile-Leu residues in the N-terminus are far apart in the
monomer, and therefore they are likely to be far apart in
the tetramer, where there is suspected b-structure (19).
There is a pair of these residues in the C-terminus (Ile94;
Leu96), but again it is likely that this sequence acts as a
monomer to bind LC3 and therefore it is unlikely to form
an Ile-Leu cross peak in the 13C correlation spectra. The
Val-Leu pair presents a possible interaction in the N-terminus if intermonomer b-strand hydrogen bonding occurs, but
even here the distance between Ca sites would be >>10 Å
for a parallel sheet structure. In the C-terminus, the Val residue (Val92) is also in the LC3 interacting region and unlikely to have intermonomer interactions with Leu96 as
described above. The fourth sample was Phe-Leu labeled,
and the only possible intermonomer interactions between
these residues are those involving the Phe residues of the
amphipathic helix, because the only terminal region Phe
residue is Phe91 in the LC3 binding site. Below we will
only discuss the ambiguities that might arise within the
conductance domain (residues 23–62) of the full-length
protein.
Proton binding
In studies of the M2FL protein in lipid bilayer preparations
the structural influences of proton, AMT, and cholesterol
binding are assessed through the use 13C-13C distance restraints between monomers. At high pH (R7.5) the four histidines (His37) responsible for proton transport are in their
neutral state, and at low pH (5.0) the proton channel is activated by having the histidine tetrad charged with a mix
of þ2 and þ3 states (42–44). The structural influence of
pH activation has been assessed by observing each of these
mixed labeled samples at pH 7.5 and 5.0 (Figs. 2 and S2)
with a spectral slice through a well-resolved resonance of
one of the two amino acid-labeled monomers. Based on
the structure of the M2CD in lipid bilayers (PDB: 2L0J)
at pH 7.5, the minimal Ca-Ca distance between Val and
Leu residues is the Val28 and Leu26 distance of 8.1 Å
(Fig. 3 A; Table 1). In the DARR spectrum at a 500 ms mixing time, a cross peak is not observed (Fig. 2 A, pH 7.5),
while at pH 5.0 this cross peak is observed. For Ala Ca
and Ile Cg1, the minimal distance in PDB: 2L0J is 3.6 Å between residues Ala30 and Ile35, respectively. This cross peak
is observed at pH 7.5, but not at pH 5.0 (Fig. 2 B). Based on
the PDB: 2L0J structure, two Ile and Leu Ca interactions
could contribute to observed cross peaks: an 8.1 Å distance
between residues Ile32 and Leu26, and a 7.7 Å distance
between residues Ile33 and Leu38. An Ile-Leu cross peak is
observed from both pH 7.5 and 5.0 samples.
The specific labels and relatively long mixing times
permit spin diffusion over relatively long distances (45)
and therefore, with the possible exception of the ambiguous
Ile-Leu restraints (Fig. 2 C), the pH 7.5 data is consistent
with the PDB: 2L0J structure of M2CD obtained at the
same pH. Despite the long distances in the PDB: 2L0J
structure for the Ile32-Leu26 and Ile33-Leu38 Ca-Ca pairs
(Fig. 3 A), the combination of a modest change in the
full-length protein structure at the helix-helix interface
and/or a slightly different tilt or rotation angle for the helix
Biophysical Journal 110, 1391–1399, March 29, 2016 1393
Ekanayake et al.
FIGURE 2 Structural influences of pH on M2FL. Spectral slices obtained at 262 K from 13C-13C DARR spectra of 13C amino acid-labeled mixed M2
samples at pH 7.5 and 5.0. (A) Spectral slices through Val-Ca frequency (66.8 ppm) using a mixing time of 500 ms and a 10.8 kHz spinning rate. (Dashed
line) Leu-Ca frequency (~58 ppm). (B) Spectral slices through Ala-Ca frequency (55.8 ppm) using a mixing time of 300 ms and a 12.2 kHz spinning rate.
(Dashed line) Ile-Cg1 frequency (~27 ppm). (C) Spectral slices through Leu-Ca frequency (58.0 ppm) using a mixing time of 500 ms and a 12.2 kHz spinning rate. (Dashed line) Ile Ca frequency (65 ppm). (D and E) Spectral slices through Leu-Cg frequency (26.6 ppm) using a mixing time of 500 ms and a
9.0 kHz spinning rate. (Dashed lines) Phe-Cg (~138 ppm) and Phe-Cdε (~130 ppm) frequencies.
at one or both of these sites could better account for the
observed cross peaks. Large aliphatic residues (L26, I33,
I35, and L38) are in the helical interface, and consequently,
a change in side-chain rotameric states for any of these residues could significantly change the helix packing and alter
FIGURE 3 Interhelical distances measured in PDB: 2L0J of the M2
conductance domain structure characterized by solid-state NMR in a lipid
bilayer environment. (A) Distances between the TM helices. (B) Distances
between the juxtamembrane helices or between the juxtamembrane helix
and the TM helix. Note in the experimental samples that the labeled Phe
and Leu are not on the same protein monomer.
1394 Biophysical Journal 110, 1391–1399, March 29, 2016
the distances by a couple of Ångstroms, accounting for the
pH 7.5 results in Fig. 2 tabulated in Table 1. Recently,
conformational heterogeneity in the neutral His37 tetrad
has been observed, and it was suggested that it could have
been induced by heterogeneity in helix packing (44). In
other words, it is not expected that a single set of rotameric
states represents the native structure at even the low temperatures used here.
Although no significant pH dependence in the N-terminal
helical structure has been observed previously from truncated constructs of the M2 protein (46), here it is clear
that a structural change does occur in the full-length protein
based on specific interhelical distance differences upon
channel activation. At pH 5.0, Val28-Leu26 are closer
together, while Ala30-Ile35 are further apart and the ambiguous Leu-Ile results remain constant; however, compensating differences between these ambiguous pairs could
occur. This structural perturbation could be the result of a
small decrease in the helical tilt or small change in the rotational orientation of the helices.
For the amphipathic helix, cross peaks are observed at
pH 7.5 between the Phe ring carbons and Leu Cg sites
(Fig. 2 D). The M2CD PDB: 2L0J structure is considered
an excellent model for this portion of the protein, because
the amphipathic helix has the tilt and rotation angle as previously characterized for the full-length protein at pH 8.0
(47). From the PDB: 2L0J structure, two possible interactions (Phe48-Leu59 and Leu46-Phe54) could contribute to
these cross peaks (Fig. 3 B). Both of these are potential sources for the observed cross-peak intensity that represents interactions between the C-terminus of the amphipathic helix
(Leu59 and Phe54) and the start of the amphipathic helix
(Phe48) or the C-terminus of the TM helix (Leu46). In the
PDB: 2L0J structure, Leu46 Cg-Phe54 Cdε distances range
from 4.6 to 7.0 Å for three of the four residue pairs in this
Structural Influences to M2FL Protein
TABLE 1
pH Dependence of Cross Peaks and Correlation with Conductance Domain Structure PDB: 2L0J
Residues Involved
Leu26Ca-Val28Ca
Ala30Ca-Ile35Cg2
Leu26Ca-Ile32Ca
Leu38Ca-Ile33Ca
Leu46Cg-Phe54Cdε
Leu59Cg-Phe48Cdε
Leu46Cg-Phe54Cg
Leu59Cg-Phe48Cg
L59V Leu46Cg-Phe54Cdε
L59V Leu46Cg-Phe54Cg
Distance Range in
PDB: 2L0J (Å)
pH 7.5 Cross Peaks
Observed
pH 7.5 Agreement with
PDB: 2L0J Distances
pH 7.5 Cross Peaks
Observed
8.1–8.2
3.6–3.9
7.5–8.1
7.7–8.2
4.6–7.0a
5.2–6.7a
6.4–7.4a
5.0–5.7a
4.6–7.0a
6.4–7.4a
—
þ
þb
þb
þc
þc
þd
þc
þ
—
þ
þ
þb
þb
þc
þc
þc
þc
þ
—
þ
—
þb
þb
þc
þc
þc
þc
—
—
a
Measured range for three out of four residue pairs in the PDB: 2L0J WT protein.
The observed interaction could be the result of a short distance for either of these two Leu/Ile pairs.
c
The observed interaction could be the result of a short distance for either of these two Leu/PheCde pairs.
d
The observed interaction could be the result of a short distance for either of these two Leu/PheCg pairs.
b
tetrameric structure; and for the Leu46 Cg-Phe54, Cg distances range from 6.4 to 7.4 Å (Table 1). In part, these
increased distances account for the weaker Phe Cg crosspeak intensity in Fig. 2, D and E. For three of the Phe48
Cdε-Leu59 Cg pairs, distances range from 5.2 to 6.7 Å
and for the Phe48 Cg-Leu59 Cg distances range from 5.0
to 5.7 Å. In Fig. 2 D, cross peaks from Leu Cg to Phe
Cdε and Phe Cg are observed at both pH 7.5 and 5.0.
When the Leu-labeled L59V M2 mutant was mixed with
Phe-labeled M2WT, a cross peak was still observed to Phe
Cdε (Phe54) from Leu46 Cg (Fig. 2 E), but at substantially
reduced intensity, suggesting that both pairs (Phe48-Leu59
and Leu46-Phe54) contribute to the strong Phe-Leu wildtype (WT) cross peaks at pH 7.5 in Fig. 2 D. However,
the Leu46 Cg-Phe54 Cg cross peak is not observed, in part
due to the weaker transfer to the nonprotonated Phe Cg
and the longer Leu46 Cg-Phe54 Cg distance (Table 1).
Hence, even this distance subtlety seems to be consistent
with the PDB: 2L0J structure. Interestingly, at pH 5.0, intensity values are observed for the WT sequences that are
similar to those seen at pH 7.5. This appears to be at odds
with electron spin resonance (ESR) results that noted
different water accessibility at low pH for spin labels on
the amphipathic helix and hence a different structural
arrangement at low pH (48).
AMT binding
The binding of AMT results in no significant differences in
the cross-peak intensities compared to protein samples at the
same pH without AMT in the N-terminus of the TM helix
(Figs. 4, A–C, and S3). This is consistent with the solid-state
NMR studies of the truncated samples of the M2 protein
(7,11). The ESR studies using spin labels tethered to
the N-terminus of the TM domain showed changes upon
AMT binding that were interpreted as pore diameter restrictions, but a change in structure of the N-terminal residue
could account for the spectral changes (49). The C-terminus
of the TM helix was not explored directly by our 13C-13C
strategy, because the helices are more splayed apart in this
portion of the structure, resulting in longer distances between the TM backbones. However, changes in the structure
induced by AMT binding in this region can be deduced from
observations of cross peaks between the TM helix and the
amphipathic helix using mixed 13C Phe and 13C Leu labeling (Fig. 4, D and E). Although at high pH, strong Phe-Leu
cross peaks were observed in the absence of AMT, these
cross peaks were not observed in the presence of the drug
(Fig. 4 D). At low pH, strong cross peaks are observed
with these labels both with and without AMT (Fig. 4 E;
Table S1). Because this distance tethers the end of the
amphipathic helix to the structural kink between the TM
and amphipathic helix, it means that there is no change in
the tilt of the C-terminal portion of the TM helix at low
pH in the presence of AMT; however, at high pH, a significant change in tilt is possible upon AMT binding, as has
been suggested by both solid-state NMR and ESR of the
M2 TMD and M2CD, respectively (7,49,50).
Cholesterol binding
When cholesterol is added to the lipid composition of the
membrane at pH 7.5, the binding of AMT has no influence
on the cross peaks between Leu and Phe (Figs. 5 and S4).
This result suggests that cholesterol is stabilizing the structure of the amphipathic helix in the lipid interface, as it is
represented in the PDB structure, 2L0J. When cholesterol
is labeled with 13C2,3,4 and mixed with 13C Phe-labeled
M2FL, strong cross peaks are observed in slices through
the cholesterol C2 resonance frequency to the ring carbons
of Phe, as shown in Fig. 6, A and B. No cross peaks were
observed in spectral slices observed through the C25 resonance of cholesterol labeled with 13C25,26,27 mixed with
13
C Phe-labeled M2FL. These results suggest a standard
orientation for cholesterol in the lipid bilayer, including
for DMPC lipids used in these experiments (51), with the
Biophysical Journal 110, 1391–1399, March 29, 2016 1395
Ekanayake et al.
FIGURE 4 Structural influences on M2FL upon AMT binding. Spectral slices obtained at 262 K from 13C-13C DARR spectra of 13C amino acid-labeled,
mixed M2 samples with and without AMT at two pH values. (A and B) Spectral slices through Val-Ca frequency (66.8 ppm) using a mixing time of 500 ms
and a 10.8 kHz spinning rate. (Dashed line) Leu-Ca frequency, ~58 ppm. (C) Spectral slices through Ile-Ca frequency (65.7 ppm) using a mixing time of
500 ms and a 12.2 kHz spinning rate. (Dashed line) Leu-Ca frequency ~58 ppm. (D and E) Spectral slices through Leu-Cg frequency (26.6 ppm) using a
mixing time of 500 ms and a 9.0 kHz spinning rate. (Dashed lines) Phe-Cg frequency ~138 ppm and Phe-Cdε frequency ~130 ppm.
hydroxyl in the bilayer interface and the hydrocarbon tail
buried in the interstices of the lipid bilayer.
A consensus recognition sequence for cholesterol binding
has long been recognized (52), originally based on studies of
a TM helix in peripheral-type benzodiazepine receptors. This
CRAC sequence (–L/V-(X)1-5-Y-(X)1-5-R/K-) was observed
as a surface from TM helix 4 of these receptors that interacted
with cholesterol, such that the Leu/Val residue was in the hydrophobic region of the bilayer and the Arg/Lys was in the
bilayer interface with a Tyr residue midway between these
sites. Subsequently, similar sequences have been recognized
in G-protein-coupled receptors (GPCRs) (53,54) and generalized to include residues lining a cleft between a pair of
TM helices that form a pocket for binding cholesterol. These
sequences are referred to as ‘‘cholesterol consensus motifs’’.
In the b-adrenergic receptor, the interfacial positively
FIGURE 5 Influence of cholesterol on AMT-bound M2FL at pH 7.5. Slices from two-dimensional DARR spectra of 13C Phe:13C Leu mixed sample
of M2FL in lipid bilayers at 262 K both with and without cholesterol and
AMT bound. Slices through Leu-Cg at 26.6 ppm. (Dashed lines) Phe Cg
(~138 ppm) and Phe Cdε (130 ppm). Spectra obtained with 500 ms mixing
time and a 9.0 kHz spinning rate.
1396 Biophysical Journal 110, 1391–1399, March 29, 2016
charged residue was shown to be in an amphipathic helix
(53), but the other residues of the CRAC or cholesterol
consensus motifs are in TM helices and not in the amphipathic helices, as has been suggested for M2 (30,55–57).
Stewart et al. (55) mutated a couple of the consensus residues of M2, without significantly influencing viral replication in vitro, suggesting that cholesterol binding may not be
important for viral budding although it had some effect on
morbidity and mortality in a mouse model. However, M2
has affinity for the periphery of the raftlike domains where
there is a considerable cholesterol lipid fraction. Indeed, M2
is recognized as facilitating viral budding from raftlike domains by inducing membrane curvature (58) in the absence
of ESCRT proteins that are not expressed by influenza viruses (32,56). Specifically, the critical role of amphipathic
helix by inducing membrane curvature during viral budding
is known (59). Furthermore, it is known that the native Cys50
residue (here C50S) in the middle of the amphipathic helix
is palmitoylated, which is often affiliated with cholesterol
binding (34). Schroeder et al. (30) showed that the M2FL
is copurified with cholesterol. Consequently, there is reason
to anticipate that M2 would bind cholesterol as a way to
attract the protein to the viral budozone, although it is also
attracted there by its affinity for the water-soluble M1 protein (31,60). Thaa et al. (57) showed that raft-targeting of
M2 was not dependent on the CRAC motif, but was dependent on palmitoylation. More recently, Thaa et al. (57) have
also shown via FRET studies that neither palmitoylation nor
the key cholesterol binding consensus residues are necessary to target M2 to the viral budozone. Consequently, it
is not clear that any of the CRAC motif residues are involved
in cholesterol binding to M2.
Four Phe residues (47, 48, 54, and 55) in the amphipathic
sequence are distributed on either side of the palmitoylation
site. Only two of these four Phe residues are oriented toward
the lipid environment, residues Phe47 and Phe54, which
would be in an appropriate position to interact with
Structural Influences to M2FL Protein
FIGURE 6 Cholesterol binding to the amphipathic helix of M2FL. (A) Structure of cholesterol -2, 3, 4- 13C- and -25, 26, 27- 13C-labeled cholesterol that
was added to 13C-Phe-labeled M2 samples. (B) DARR spectral slices through C3 and C25 of cholesterol, with 500 ms mixing and a 9 kHz spinning rate. (C)
Likely cholesterol binding site modeled on the M2 CD structure (PDB: 2L0J).
cholesterol and form cross peaks with the C2 carbon of
cholesterol. Note that these residues are in an I-to-Iþ7
sequential arrangement so that they are on the same surface
of the helix. Moreover, the PDB: 2L0J structure suggests
that Phe54 may be in a more appropriate position for this
interaction than Phe47, for several reasons. First, the
carbonyl group of Ser50 is the most exposed carbonyl on
this surface of the amphipathic helix as a result, in part, of
the small Ser (Cys) sidechain (Fig. 6 C). This would be an
ideal location for hydrogen bonding of the cholesterol hydroxyl to the protein. Second, this would position cholesterol in a cleft between the C-terminal TM helices and be
flanked in the native protein by the palmitoyl group linked
to the Cys50 residue. In this position, as shown in the structural model in Fig. 6 C, the Phe54 aromatic ring would be in
van der Waals contact with cholesterol. Indeed, the orientation of the cholesterol and palmitoyl group to the amphipathic helix in the b2-adrenergic GPCR (61) appears to be
similar to what is modeled here and for which we have
observed very strong 13C Phe-13C3 cholesterol cross peaks
consistent with close contact in the structural model with
Phe54.
Above, it was noted that at pH 5.0 the binding of AMT did
not significantly influence the protein structure as assessed by
the Phe/Leu cross peak (Fig. 4 E), but at pH 7.5 the binding of
AMT resulted in the loss of the Phe/Leu cross peak. The addition of cholesterol to the pH 7.5 preparation with AMT prevents the loss of the Phe/Leu cross peaks as shown in Fig. 5.
Consequently, cholesterol appears to stabilize the amphipathic helix structure in the membrane interface. While this
stabilizing effect of cholesterol was not required in the
absence of drug, its presence can be assumed from the multiple potential interactions between cholesterol and the amphipathic helix (hydrogen bonding directly or indirectly with
Ser50 (Cys50) and van der Waals interactions with Phe54) as
well as interactions with the C-terminal region of the TM
helices and with the palmitoyl group of Cys50 (van der Waals
interactions). These interactions could result in considerable
stabilization of the amphipathic helix position in the lipid
interface, and potentially this is important when the protein
is inducing substantial curvature strain on the membrane in
the budozone upon membrane fusion in the endosome.
CONCLUSIONS
Numerous specific structural restraints have been observed
here using mixed 13C-labeled M2FL monomers in the tetrameric channel. These restraints have characterized pHdependent structural perturbations in the N-terminal region
of the TM helices that had not been observed in prior studies
of truncated protein constructs in various detergent-based or
lipid-based environments. Other structural perturbations
that had been observed in the C-terminal region of the TM
domain were not confirmed in the spectra of M2FL, suggesting increased stability as a result of the presence of the
C-terminal domain. NMR interhelical distance restraints
between the amphipathic and TM helices had not been
observed previously; here the restraints provided by both
Leu59-Phe48 and Leu46-Phe54 are very significant for characterizing the M2 structure at the inner membrane interface of
the viral capsid. These restraints document the proximity of
the C-terminal end of the amphipathic helix with the C-terminal end of the TM helix in the adjacent monomer. When
AMT was bound at pH 7.5, these Leu-Phe interactions were
lost, suggesting a significant structural change.
Cholesterol binding to M2 has been demonstrated, and
that it binds in a typical cholesterol binding site and orientation, one that utilizes the amphipathic helix where it interacts with one of the Phe side chains, mostly likely Phe54,
and it may hydrogen-bond with the Ser(Cys)50 carbonyl
oxygen. In addition, there is an interhelical crevice in the
PDB: 2L0J structure into which cholesterol can optimally
form van der Waals interactions. This crevice represents a
weak point in the M2 structure with a relatively large separation of the helices. It is important for proteins to avoid
fenestrations into the fatty acyl environment that could
lead to fatty acyl penetration into the pore, thereby disrupting the functional mechanism. In the native protein, interactions with the Cys50 palmitoyl group could further
stabilize cholesterol binding. The binding of cholesterol
Biophysical Journal 110, 1391–1399, March 29, 2016 1397
Ekanayake et al.
to the amphipathic helix and stabilization of this conformation has important consequences for the functionality of the
protein. Influenza A does not have the ESCRT proteins that
facilitate viral budding for many viruses (60). Instead, M2
facilitates membrane curvature through the position of
its amphipathic helix, generating a dramatic pyramidal
shape that induces considerable membrane curvature. If
the amphipathic helix changes position, such as in the formation of a water-soluble, four-helix bundle (e.g., the solution NMR M2 structure, PDB: 2RLF (8)), it eliminates
M2’s ability to induce curvature. The virus buds out from
a raftlike domain and the relatively short hydrophobic
dimension of the M2 TM helices largely restricts M2 to
the thinner surrounding membrane domain rather than the
raftlike domain. Consequently, M2 clusters at the periphery
of the raftlike domain where cholesterol is present in high
concentration, thereby facilitating the binding of cholesterol to M2 and thus stabilizing the amphipathic helix in
the lipid interface-inducing curvature of the host membrane
needed for viral budding from the host cell. A similar process may occur when the viral interior becomes acidified in
the endosome before the fusion of the viral membranes
with the endosomal membrane.
SUPPORTING MATERIAL
Supporting Materials and Methods, four figures, and one table
are available at http://www.biophysj.org/biophysj/supplemental/S00063495(16)00152-1.
AUTHOR CONTRIBUTIONS
E.V.E. and T.A.C. designed the research; E.V.E. expressed the protein and
prepared the samples; and E.V.E. and R.F. conducted the NMR experiments
and analyzed the data. All authors contributed to the writing of the article.
ACKNOWLEDGMENTS
The authors thank W.W. Brey, P. Gor’kov, J. Kitchen, and S. Rainer at the
National High Magnetic Field Laboratory for valuable assistance with
probe technology; Z. Gan and I. Hung for spectrometer help; J. Dai for
computational modeling; and H. Qin and C. Escobar for help cloning and
optimizing protein expression and purification.
The work was made possible by a grant from the National Institutes of
Health (No. AI R01-023007), and the NMR experiments were performed
at the National High Magnetic Field Laboratory supported by National Science Foundation Cooperative Agreement No. DMR-1157490 and the State
of Florida.
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Biophysical Journal 110, 1391–1399, March 29, 2016 1399
Biophysical Journal, Volume 110
Supplemental Information
Structural Influences: Cholesterol, Drug, and Proton Binding to FullLength Influenza A M2 Protein
E. Vindana Ekanayake, Riqiang Fu, and Timothy A. Cross
Supporting Information
Structural Influences of Cholesterol, Drug & Proton Binding to the Full Length Influenza AM2
Protein
E. V. Ekanayake, R. Fu, T. A. Cross
Material and Methods
•
Expression of M2 full-length
Protein was expressed with N-terminal Histidine-6 tag similar to previously reports1.
Briefly, a single colony of BL21 (DE3) RP codon plus cells from a transformation plate were
used to inoculate two 50 ml aliquots of LB media with 100 µg/ml ampicillin antibiotics. This
culture was grown at 37 °C overnight (< 14 h) in a shaker incubator (250 rpm) and transferred
into 1L of LB media with 100 µg/ml of ampicillin. These cells were allowed to grow at 37 °C till
an OD600 value of 0.7 (1 cm pathlength) was reached. The cells were centrifuged at 4000 g for
10 mins at room temperature.
Any residual LB was washed away by re-suspending the
combined pellet (from 2 L LB) in 50 mL of M9 salt followed by another round of centrifugation.
The pelleted cells were re-suspended in 1 L of M9 salts supplemented with 100 mg of the 13Clabeled L-amino acid of interest (Cambridge Isotope Labs, Sigma–Aldrich) and unlabeled amino
acids for all remaining amino acids. Then this mixture was incubated at 37 °C for 40 min in the
same shaker, before adding isopropyl-β-D-thiogalactopyranoside (IPTG) to a final concentration
of 0.4 mM to induce protein overexpression. The culture was incubated for an additional 4 h at
22°C before cells were harvested by centrifugation at 5000 g for 10 mins at room temperature.
1 Then the pellet was re-suspended in 30 mL of buffer containing 20mM Tris pH 8.0, 500 mM
NaCl and 15% glycerol and stored at -80 ºC.
•
Purification of M2 full-length
The frozen bacterial cell suspension at -80°C was thawed and treated at room
temperature. Then Benzonase nuclease (Novagene; 4µL of 250 U/µl) was added to the cell
culture to degrade nucleic acids (DNA, RNA). To help disrupt the bacterial cell-walls 0.25mg of
lysozyme (Sigma-Aldrich)/L of cell culture was added to the cell suspension at room
temperature and all components were mixed using an orbital shaker at room temperature for 1 h
prior to cooling the suspension on ice for ~ 1 h. Mechanical cell disruption (French Press;
Thermo Scientific) was used to lyse cells. Cells were subjected to French Press three times at
12,000 PSI. Then, Empigen-BB (Sigma-Aldrich- 35% active substance in H2O) was added to the
resulting lysate to solubilize the cell membranes until the concentration becomes 3% (wt/vol).
The suspension was rotated gently using a tube shaker (Thermo Scientific Labquake) overnight
(~14 h) at 4 °C. The resulting membrane solubilized cell lysate was subjected to
ultracentrifugation at 228,000 g for 45 min at 8 ºC to remove insoluble cell debris as the proteins
(membrane and soluble) remained in the supernatant. Nickel affinity column chromatography
was used for purification. A 5 ml HisTrap HP Nickel Column (GE Healthcare Life Sciences) was
pre-packed with Ni Sepharose High Performance resin. The column was equilibrated with the
equilibration buffer (500mM NaCl, 20mM Tris pH 8.0, 1% wt/vol Empigen-BB, 20mM
imidazole, 15% glycerol) using an ÄKTAxpress (GE Healthcare Life Sciences) protein
purification system and the protein supernatant was loaded onto this column. The column was
washed with equilibration buffer (until UV280 absorbance returned to baseline) and subsequently,
washed with wash 1 buffer (500mM NaCl, 20mM Tris, pH 8.0, 0.7% wt/vol Empigen-BB, 60
2 mM imidazole, 15% glycerol). The detergent was exchanged using five column volumes (20
mL) of exchange buffer [500mM NaCl, 20mM Tris, pH 8.0, 0.5% wt/vol octyl-β-Dglucopyranoside (OG), 15% glycerol]. Finally, the protein was eluted using elution buffer
(500mM NaCl, 20mM Tris, pH 8.0, 0.5% wt/vol OG, 250 mM imidazole, 15% glycerol)
resulting in ~ 35-40 mg of protein for 1 L of M9 media. To break any disulphide bonds that
might be present in the N-terminus, the purified protein was treated with 5 mM dithiothreitol
(DTT-from Sigma-Aldrich), so that different isotopically labeled monomers from different
cultures would blend properly in the next step.
•
Preparation of distinct isotopically labeled mixture, reconstitution into lipid bilayers
and MAS sample preparation
Different isotopically labeled monomers are mixed 1:1 (mol/mol), in a small round-
bottomed flask and bath sonicated (Branson) for ~ 5 mins to make sure the mixture was
optimally blended. A 4:1 molar ratio of dimyristolphosphatidylcholine (DMPC) and
dimyristolphosphatidylglycerol (DMPG) lipid (Avanti Polar Lipids) was used to reconstitute the
protein in a1:30 protein to lipid molar ratio. First, lipid mixture was re-suspended in 40 0C Tris,
pH 8.0 buffer (1 mL for every 10 mg of lipid mixture) and subjected to vortex mixing (VWR) to
obtain homogenized suspension. Then OG was added from a stock solution (~ 15% wt/vol) to a
final OG concentration is 3% (wt/vol). Once the lipid mixture becomes clear due to the
formation of mixed micelles, the mixture was transferred to a FalconTM 50mL conical tube
(Fisher Scientific) and kept in an orbital shaker for 30 mins at room temperature for completion
of lipids solubilization. The protein mixture was added to the lipid mixture in the orbital shaker
and mixed for an aditional ~ 3 hours at room temperature. When cholesterol was added (SigmaAldrich) a molar ratio of 1:6 (cholesterol:other lipids) was used. The protein-lipid mixture was
transferred to a pre-soaked dialysis bag (SpectraPor) having a molecular weight cut-off of 3.5
3 kDa, and dialyzed against 2 L of buffer (20 mM HEPES for pH 7.5 or 20 mM citrate buffer for
pH 5.0) at 37 0C to remove detergent. The external buffer was changed twice a day and typically
carried out for four days. Removal of detergent was confirmed by HPLC using evaporative light
scattering detection (ELSD)2. For amantadine binding experiments, the drug was added after the
removal of detergents to the resulting proteoliposome suspension at a 1:2 molar ratio of
channel:drug. The resulting proteoliposome preparation was bath sonicated for ~5 min to make
the liposomes homogeneous followed by ultracentrifugation at 354,000 g at 8 °C for 5 h. Then
this pellet was transferred to a 98% hydration chamber containing saturated potassium sulfate
until the excess buffer is removed.
•
Magic Angle Spinning NMR experiments
M2 proteoliposomes were packed into 3.2 mm thin walled MAS rotors (Revolution
NMR) as reported3 (Das et al., 2013). The ssNMR experiments were acquired on a Bruker
Avance 600.1 MHz (14.1 T) NMR spectrometer at 262 K. Home built Low-E triple resonance
probes, which minimize rf-induced sample heating, were utilized4,5 for all experiments. Different
mixing times (300-500 ms) were used for the
13
C-13C dipolar assisted rotational resonance
(DARR) experiments. Spinning rates from 9 to 12.2 kHz were used for different samples to
avoid spinning sideband overlap with the potential crosspeaks. 200 to 512 t1 increments were
used for the DAAR experiments with 224-512 transients for each t1 point. Topspin (Bruker) was
used to process all data usig Gaussian window functions (LB=-50 Hz and GB=0.12) in both
dimensions. All 13C chemical shifts were externally referenced using 13C-CO labeled glycine and
setting the carbonyl carbon resonance to 178.4 ppm relative to TMS.
4 Supplementary Figures and Tables
Figure S1: 2D DARR spectra of M2FL with individually 13C labeled amino acids. Sample pH is
at 7.5 and the temperature is 262 K. MAS frequencies: 12.2 kHz for Ile, Leu; 10.8 kHz for Val;
9.0 kHz for Phe. Natural abundant 13C lipid signals appear at 16 ppm, 27 ppm and 35 ppm. Note
multiple crosspeaks, between lipid C2 at ~ 35ppm to amino acid residues, in all spectra can be
observed. Amino acid residues located in the helical interface can be expected to contribute to
these signals.
5 Figure S2. Structural influences of pH on M2FL. Spectral slices obtained at 262K from 13C-13C DARR
spectra of 13C amino acid labeled mixed M2 samples at pH 7.5 and 5.0. A) Spectral slices through Val-Cα
frequency (66.8 ppm) using a mixing time of 500 ms and 10.8 kHz spinning rate. Dashed line is at LeuCα frequency (~58 ppm) B) Spectral slices through Ala-Cα frequency (55.8 ppm) using a mixing time of
300 ms and 12.2 kHz spinning rate. Dashed line is at the Ile-Cγ1 frequency (~27 ppm) C) Spectral slices
through Leu-Cα frequency (58.0 ppm) using a mixing time of 500 ms and 12.2 kHz spinning rate. Dashed
line is at Ile Cα frequency (65 ppm). D & E) Spectral slices through Leu-Cγ frequency (26.6 ppm) using a
mixing time of 500 ms and 9.0 kHz spinning rate. Dashed lines are at Phe-Cγ (~138 ppm) and Phe-Cδε
(~130 ppm) frequencies.
6 Figure S3. Structural influences on M2FL upon AMT binding. Spectral slices obtained at 262K from 13C13
C DARR spectra of 13C amino acid labeled mixed M2 samples with and without AMT at two pH values.
A & B) Spectral slices through Val-Cα frequency (66.8 ppm) using a mixing time of 500 ms and 10.8
kHz spinning rate. Dashed line at Leu-Cα frequency, ~58 ppm. C) Spectral slices through IleCα frequency (65.7 ppm) using a mixing time of 500 ms and 12.2 kHz spinning rate. Dashed line at LeuCα frequency ~58 ppm. D & E) Spectral slices through Leu-Cγ frequency (26.6 ppm) using a mixing time
of 500 ms and 9.0 kHz spinning rate. Dashed lines are at Phe-Cγ frequency ~138 ppm and Phe-Cδε
frequency ~130 ppm.
7 Figure S4: Influence of cholesterol on AMT bound M2FL at pH 7.5. Slices from 2D DARR spectra of
13
C Phe:13C Leu mixed sample of M2FL in lipid bilayers at 262K with and without cholesterol and AMT
bound. Slices through Leu-Cγ at 26.6 ppm. Dashed lines are at Phe Cγ (~138 ppm) and Phe Cδε(130
ppm). Spectra obtained with 500 ms mixing time and 9.0 kHz spinning rate.
8 Table S1. Influence of AMT binding to the observation of intermonomer distance
crosspeaks.
Residues
pH
Involved
Without
AMT
With
AMT
2L0J (Å)
(pH 7.5)
Crosspeaks
Observed
Crosspeaks
Observed
8.1-8.2
+
+
+
+
+
-
+
+
+
-
+
+
Leu26Cα-Val28Cα
7.5
Leu26Cα-Val28Cα
5.0
Leu26Cα-Ile32Cα
7.5
7.5-8.1
Leu38Cα-Ile33Cα
7.5
7.7-8.2
Leu46Cγ-Phe54Cδε
7.5
4.6-7.0*
Leu59Cγ-Phe48Cδε
7.5
5.2-6.7*
LeuCγ-PheCδε
5.0
Leu46Cγ-Phe54Cγ
7.5
6.4-7.4*
Leu59Cγ-Phe48Cγ
7.5
5.0-5.7*
LeuCγ-PheCγ
5.0
* Measured range for three of four residue pairs in 2L0J
Supplementary References
(1)
Miao, Y. M.; Qin, H. J.; Fu, R. Q.; Sharma, M.; Can, T. V.; Hung, I.; Luca, S.;
Gor'kov, P. L.; Brey, W. W.; Cross, T. A. Angew. Chem. Int. Edit. 2012, 51, 8383.
(2)
Mourey, T. H.; Oppenheimer, L. E. Anal. Chem. 1984, 56, 2427.
(3)
Das, N.; Murray, D. T.; Cross, T. A. Nat. Protoc. 2013, 8, 2256.
(4)
Gor'kov, P. L.; Chekmenev, E. Y.; Li, C. G.; Cotten, M.; Buffy, J. J.; Traaseth, N.
J.; Veglia, G.; Brey, W. W. J. Magn. Reson. 2007, 185, 77.
9 (5)
McNeill, S. A.; Gor'kov, P. L.; Shetty, K.; Brey, W. W.; Long, J. R. J. Magn.
Reson. 2009, 197, 135.
10