dissection of antibacterial and toxic activity of melittin ... · neeta asthana‡, sharada prasad...
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Dissection of antibacterial and toxic activity of melittin: A leucine zipper motif plays
crucial role in determining its hemolytic activity but not antibacterial activity#
Neeta Asthana‡, Sharada Prasad Yadav‡ and Jimut Kanti Ghosh*
Molecular and Structural Biology Division
Central Drug Research Institute
Lucknow 226 001
India ‡Authors contributed equally to this work
Running Title: Leucine zipper motif in a naturally occurring antimicrobial peptide
*To whom correspondence should be addressed
Telephone: 2212412 (Ext. 4282)
Fax: 091-522-223405/223938
E-mail: [email protected] #CDRI communication No. 6622
JBC Papers in Press. Published on October 8, 2004 as Manuscript M408881200
Copyright 2004 by The American Society for Biochemistry and Molecular Biology, Inc.
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Leucine zipper motif in a naturally occurring antimicrobial peptide 2
Summary:
Melittin, a naturally occurring antimicrobial peptide, exhibits strong lytic activity
against both eukaryotic and prokaryotic cells. Despite a tremendous amount of work
done, very little is known about the amino acid sequence, which regulates its toxic
activity. With a goal to understand the basis of toxic activity and poor cell selectivity in
melittin, a leucine zipper motif has been identified. To evaluate the possible structural
and functional roles of this motif, melittin and its two analogs after substituting the
heptadic leucine by alanine were synthesized and characterized. Functional studies
indicated that alanine substitution in the leucine zipper motif resulted in a drastic
reduction of the hemolytic activity of melittin. However, interestingly, both the designed
analogs exhibited comparable antibacterial activity to melittin. Mutations caused
significant decrease in the membrane permeability of melittin in only zwitterionic but not
in negatively charged lipid vesicles. Although both the analogs exhibited similar
secondary structures in the presence of negatively charged lipid vesicles as melittin, they
failed to adopt significant helical structure in the presence of zwitterionic lipid vesicles.
Results suggest that the substitution of heptadic leucine by alanine impaired the assembly
of melittin in aqueous environment and its localization only in zwitterionic but not in
negatively charged membrane. Altogether, the results suggest the identification of a
structural element in melittin, which probably plays a prominent role in regulating its
toxicity but not antibacterial activity. The results indicate that probably cell selectivity in
some antimicrobial peptides can be introduced by modulating their assembly in aqueous
environment.
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Leucine zipper motif in a naturally occurring antimicrobial peptide 3
Introduction: Melittin, the major component of European bee venom, Apis mellifera is a
well-studied peptide, which is known for its strong cytolytic and antimicrobial activities
(1,2). It belongs to the family of cytolytic peptides whose members are believed to be the
key components of defense and offense mechanisms of all organisms (3,4).
A considerable number of structure-function studies have been carried out to
understand the molecular mechanism of melittin’s hemolytic and antimicrobial activities.
Replacement of the first twenty amino acids by another helix forming sequence did not
alter antimicrobial and hemolytic activity of melittin (5). However, the deletion of L (6),
K (7), V (8), L (9), L (13), L (16), I (17), W (19) and I (20) dramatically reduced
melittin’s hemolytic activity and to a lesser extent its antimicrobial activity also (6). The
crucial roles of both termini of melittin in its functional properties have been
demonstrated by the design of several analogs with deletion of N- (7,8) and C-terminal
(9) sequences.
Several efforts have been made to design analogs of melittin with decreased
hemolytic but similar antimicrobial activity. For example, the diastereomers of melittin
having D-amino acid analogs (10) at a few positions, a hybrid peptide consisting of
cecropin A and melittin sequences (11) and an analog designed from the C-terminal 15-
residue fragment (12) exhibited similar antibacterial activity to melittin but much less
hemolytic activity.
The crystal structure of melittin has been solved, which indicated a tetrameric
helix-bend-helix structure (13) with two helical segments, positioned between amino acid
residues 1-10 and 13-26 and the bend region within residues 11-12.
Biophysical properties of melittin like its self-association and phospholipid
membrane-interaction have been studied extensively in order to understand its mode of
action (14-17). Although in water melittin exists as monomer with mostly random coil
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Leucine zipper motif in a naturally occurring antimicrobial peptide 4
conformation, an increase in peptide concentration, or addition of salt results the
transformation of monomer to tetramer with pronounced helical structure (18-20).
Melittin has been used as a model for studying the general features of lipid interaction of
membrane proteins. Membrane-interaction studies suggest that melittin forms
transmembrane pores in zwitterionic lipid bilayers by a barrel-stave mechanism (21-24)
and in negatively charged lipid vesicles it acts like a detergent by carpet mechanism
(25,24).
Despite a lot of work done on melittin, it is not yet clear: i) why melittin has very
low cell selectivity; i.e., why it exerts both high antibacterial and toxic activities, ii) is the
same amino acid sequence responsible for both the hemolytic and antibacterial activities?
Towards the identification of important structural and functional elements in melittin and
to gain insight about the regulation of cell selectivity in an antimicrobial peptide in
general, we have found a leucine zipper motif, located within the residues 6-20, which
has not been reported earlier to the best of our knowledge. Since this motif is known to
play important roles in the assembly of DNA-binding proteins (26,27) or membrane-
associated viral fusion proteins (28-30), it was of interest to look into the possible roles of
this motif in the structural integrity and functional activity of melittin. For this purpose,
two analogs of melittin were designed by selectively replacing heptadic leucine with
single and double alanine and synthesized. Hemolytic activity of single (MM-1) and
double alanine mutant (MM-2) against human red blood cells was significantly less than
that of melittin. Interestingly, MM-1 and MM-2 exhibited comparable antibacterial
activity to melittin. Both the designed analogs displayed contrasting membrane
permeability in zwitterionic and negatively charged lipid vesicles. The results have been
discussed in terms of the role of this motif in determining the hemolytic and antimicrobial
activity of melittin.
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Experimental Procedures:
Materials:
Rink amide MBHA resin (loading capacity, 0.63 mmol/g) and all the N-α Fmoc and side-
chain protected amino acids were purchased from Novabiochem, Switzerland. Coupling
reagents for peptide synthesis like 1-hydroxybenzotriazole (HOBT), N, N′-di-
isopropylcarbodiimide (DIC), 1,1,3,3-tetramethyluronium tetrafluoroborate (TBTU) and
N, N′-diisopropylethylamine (DIPEA) were purchased from Sigma, USA.
Dichloromethane, N, N′ dimethylformamide (DMF) and piperidine were of standard
grades and procured from reputed local companies. Acetonitrile (HPLC grade) was
procured from Merck, India while trifluoroacetic acid (TFA) was purchased from Sigma.
Egg phosphatidylcholine (PC), egg phosphatidylglycerol (PG) and dimyristoyl
phosphatidylethanolamine (PE) were procured from Northern Lipids Inc., Canada while
cholesterol (Chol) was purchased from Sigma. 3,3′-Dipropylthiadicarbocyanine iodide
(diS-C3-5) and NBD-fluoride (4-fluoro-7-nitrobenz-2-oxa-1, 3-diazole) were purchased
from Molecular probes (Eugene, OR). Rests of the reagents were of analytical grade and
procured locally; buffers were prepared in milli Q water.
Peptide Synthesis, Fluorescent labeling and Purification: All the peptides were
synthesized manually on solid phase. Stepwise solid phase syntheses were carried out on
rink amide MBHA resin (0.15 mmole) utilizing the standard Fmoc chemistry, employing
DIC/HOBT or TBTU/HOBT/DIPEA coupling procedure (30-32) with checking of de-
protection of α-amino group and the coupling of amino acids by usual procedures
(33,34). After the synthesis was over, each peptide was cleaved from the resin by a
standard method as reported earlier (32) and precipitated with dry ether. Labeling at the
N-terminus of peptides with a fluorescent probe was achieved by a standard procedure
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(35,36,30,32) and after sufficient labeling; the labeled peptides were cleaved from the
resins and precipitated. All the peptides were purified by RP-HPLC on an analytical
Vydac C18 column using a linear gradient of 0-80% acetonitrile in 45 min with a flow
rate of 0.6 ml/min. Both acetonitrile and water contained 0.05% TFA. The purified
peptides were ~95% homogeneous as shown by HPLC. Experimental molecular mass of
the peptides, detected by ES-MS analysis, corresponded to the desired values within 0.5
Da.
Preparation of Small Unilamellar Vesicles (SUVs): SUVs were prepared by a standard
procedure (35,36,32) with required amounts of either of the PC/cholesterol (8:1 w/w),
PC/PG/cholesterol (4:4:1 w/w) or PE/PG (7:3 w/w). Dry lipids were dissolved in
CHCl3/MeOH (2:1 v/v) in small glass vial. Solvents were evaporated under a stream of
nitrogen resulting the formation of a thin film on the wall of a glass vessel. The thin film
was re-suspended in a buffer at a concentration of 8.2 mg/ml by vortex mixing. The lipid
dispersions were then sonicated in a bath-type sonicator (Laboratory Supplies Company,
New York) for 10-20 min until it became transparent. The lipid concentration was
determined by phosphorus estimation (37).
Circular Dichroism Experiments: The circular dichroism (CD) spectra of peptides were
recorded in phosphate buffered saline (PBS, pH 7.4), and in the presence of phospholipid
vesicles by utilizing a Jasco J-810 spectropolarimeter, calibrated routinely with 10-
camphor sulphonic acid. The samples were scanned at room temperature (~300 C) in a
capped quartz cuvette of 0.20 cm path length in the wavelength range of 250-195 nm.
The fractional helicities were calculated with the help of mean residue ellipticity values at
222 nm by the following equation (38,39) as already reported (30,32). [θ] 222 − [θ]0
222 Fh =[θ]100
222 − [θ]0222
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Where [θ]222 was the experimentally observed mean residue ellipticity at 222 nm. The
values for [θ]100222 and [θ]0
222 that correspond to 100 and 0% helix contents were
considered to have mean residue ellipticity values of –32,000 and –2,000 respectively at
222 nm (39).
Membrane Permeability Assay: Dissipation of Diffusion potential from the small
unilamellar lipid vesicles: The ability of melittin and its analogs to destabilize the
phospholipid bilayer was detected by the assay of peptide induced dissipation of diffusion
potential across the lipid vesicles by employing a standard procedure (40,41) as reported
before (41,30, 32). Lipid vesicles, prepared in K+ buffer (50 mM K2SO4/25 mM HEPES-
sulfate, pH 6.8), were mixed with isotonic (K+-free) Na+-buffer followed by the addition
of the potential sensitive dye diS-C3-5. After the addition of valinomycin to the vesicle
suspension when fluorescence of the dye exhibited a steady level, the respective peptide
was added. The peptide-induced dissipation of diffusion potential as detected by an
increase in fluorescence (at 670 nm with excitation at 620 nm), was measured in terms of
percentage of fluorescence recovery (Ft), defined by:
Ft = [(It - I0)/(If - I0)] x 100 %
Where It = fluorescence after the addition of a peptide at time t, I0 = fluorescence after the
addition of valinomycin and If = the total fluorescence observed before the addition of
valinomycin.
Enzymatic Cleavage Experiments: In order to detect the location of the peptides in
their membrane-bound states, enzymatic cleavage experiments were performed with their
NBD-labeled versions as reported earlier (30,32). In brief, lipid vesicles made of either
PC/Chol, PC/PG/Chol or PE/PG were first added to the NBD-labeled peptide. When all
the peptide was bound to lipid as detected by the saturation of the fluorescence level,
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proteinase-k (final concentration, 12.0 µg/ml) was added. In this experiment
fluorescence of NBD-labeled peptide was recorded at 527 nm with respect to time (in
sec) with excitation wavelength set at 467 nm.
Hemolytic activity assay: Hemolytic activity of melittin and its analogs was assayed by
a standard procedure (10,12). In brief, fresh human red blood cells (hRBCs) that were
collected in the presence of an anti-coagulant from a healthy volunteer were washed three
times in PBS. Peptides, dissolved in water, were added to the suspension of red blood
cells (6% final in v/v) in PBS to the final volume of 200 µL and incubated at 37 0C for 35
min. The samples were then centrifuged for 10 min at 2000 r.p.m. and the release of
hemoglobin was monitored by measuring the absorbance (Asample) of the supernatant at
540 nm. For negative and positive controls hRBCs in PBS (Ablank) and in 0.2% (final
concentration v/v) Triton X-100 (Atriton) were used respectively. The percentage of
hemolysis was calculated according to the following equation.
Percentage of hemolysis = [(Asample-Ablank)/(Atriton-Ablank)] x 100
Assay of antibacterial activity of the peptides: The antibacterial activity of the peptides
was assayed in LB medium under aerobic conditions (10,12,42). Different
concentrations of each of the peptides, dissolved in water, were added in duplicate to 100
µL (final volume) of medium containing the inocula of the test organism (~106 CFU) in
mid-logarithmic phase of growth. The inhibition of growth of microorganisms was
assessed by measuring the absorbance at 492 nm after an incubation of 18-20 h at 37 0C.
The antibacterial activity of the peptides was expressed as the minimal inhibitory
concentration (MIC), the concentration at which 100% inhibition of microbial growth
was observed after 18-20 h of incubation. The microorganisms used were Gram-positive
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bacteria, Bacillus subtilis and Staphylococcus aureus and Gram-negative bacterium,
Escherichia coli.
Results:
Design of analogs of melittin: Melittin has an interesting sequence with 26 amino acids
including five cationic residues. A leucine zipper motif, with every seventh amino acid
as leucine/isoleucine (Leucine at position 6 and 13 and isoleucine at position 20) and also
leucine in two ‘d’ positions, was identified (Fig.1, panel A and B). In order to look into
the possible role of this important structural motif, two analogs of melittin were designed.
In one of which leucine at position 13 (MM-1) was replaced with alanine and in the other
(MM-2) leucine at positions 6 and 13 were replaced with two alanines (Fig. 1, panel A).
Alanine was chosen for its helix propensity and similar hydrophobicity to leucine so that
the amphipathic character of melittin is retained in MM-1 and MM-2.
Mutation in the leucine zipper motif drastically reduced the hemolytic activity of
melittin: In order to look into the role of the identified leucine zipper motif in the
functional activities of melittin, hemolytic activity of melittin and its designed analogs
were assayed. The substitution of leucine with alanine resulted in a drastic reduction of
hemolytic activity of melittin against human red blood cells (hRBCs) (Fig. 2). MM-1
exhibited 10-20 % hemolytic activity of melittin while MM-2 showed around 1-2 %
activity. The results indicate that substitution of heptadic amino acids by alanine
markedly inhibited the hemolytic activity of melittin probably suggesting a role of this
motif in maintaining the lytic activity of the peptides towards the RBCs.
The designed analogs exhibited comparable anti-bacterial activity to melittin:
Melittin and its analogs were tested for growth inhibiting activity in liquid cultures of two
Gram-positive and one Gram-negative bacteria. Tetracycline was used as a positive
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control. In contrast to the hemolytic activity, the designed analogs of melittin (MM-1 and
MM-2) exhibited very similar antibacterial activities to that of melittin (Table-1). Thus
the results clearly indicate that substitution of leucine by alanine in the heptadic positions
did not affect the antibacterial activity of melittin unlike its hemolytic activity.
Contrasting difference in membrane permeability of the designed analogs of
melittin in zwitterionic and negatively charged membrane: The ability of melittin to
destabilize the phospholipid bilayer is believed to be associated with its mechanism of
action. Therefore, to understand the basis of decreased hemolytic activity and almost
unaltered antibacterial activity of the designed melittin analogs, membrane permeability
of melittin, MM-1 and MM-2 were examined by determining their efficacy to dissipate
the diffusion potential across the phospholipid vesicles with different lipid composition.
As shown in Fig. 3, panel A, membrane permeability (expressed as the percentage of
fluorescence recovery), of MM-1 was significantly lower than melittin in zwitterionic
PC/Chol vesicles, which decreased further with MM-2. However, interestingly, both
MM-1 and MM-2 exhibited very similar membrane permeability to that of melittin in
negatively charged PC/PG/Chol lipid vesicles (Fig. 3, panel B). Experimental profiles
for each of the peptides in both kinds of membranes are shown in the insets of panel A
and B of Fig. 3. Moreover, the designed analogs induced membrane permeation as
efficiently as melittin (Figure not shown) in another kind of negatively charged
membrane namely PE/PG (7:3 w/w). The results indicate that although the substitution
of leucine by alanine appreciably impaired the membrane permeability of melittin in
zwitterionic membrane, it had almost negligible effect in negatively charged membrane.
The designed analogs exhibited similar secondary structures to melittin in
negatively charged membrane but not in zwitterionic membrane: Circular dichroism
experiments were performed in order to determine the secondary structures of melittin
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Leucine zipper motif in a naturally occurring antimicrobial peptide 11
and its analogs in aqueous environment (phosphate buffered saline, PBS, pH 7.4) and in
the presence of zwitterionic and negatively charged phospholipid vesicles. The
corresponding mean residue ellipticity values of the individual peptides at 222 nm were
used to determine their helical contents at a particular environment. Although all three
peptides exhibited low helical structure in PBS (Fig. 4, panel A), the extent of helicity
was slightly more in melittin (~15%) than MM-1 (~8.5%) and MM-2 (~6.5%). However,
in the presence of increasing amount of negatively charged PC/PG/Chol lipid vesicles
significant amount of helicity was induced in all three peptides as shown by a
representative CD spectrum for each of these peptides with a fixed lipid/ peptide (L/P)
molar ratio (~32) (Fig. 4, panel B). Melittin (69%), MM-1 (67.5%) and MM-2 (68.5%)
exhibited very similar extent of helical structures indicating that the substitution of
leucine by alanine did not affect the secondary structure of melittin in the presence of
negatively charged lipid vesicles.
However, very contrasting results were obtained in the presence of zwitterionic
PC/Chol lipid vesicles (Fig. 4, panel C). Although melittin adopted a significant amount
of helical structure (47% at L/P ~24) in the presence of PC/Chol lipid vesicles, the extent
of induction of helicity was much less in MM-1 (~15%) and MM-2 (8.5%) with the same
amount of lipid vesicles.
Mutation in the leucine zipper motif affected the localization of melittin in
zwitterionic membrane but not in negatively charged membrane: In order to detect
the location of melittin and its analogs onto the membrane, proteolytic cleavage
experiments were performed with NBD-labeled peptides in their membrane-bound states.
The basis of this experiment is that NBD-labeled peptides, bound onto the membrane-
surface, are easily cleaved by a proteolytic enzyme like proteinase-k, which can be
monitored by the decrease in NBD-fluorescence from its characteristic membrane-bound
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level. On the other hand, a membrane-inserted peptide will not be accessible to
proteinase-k and therefore its NBD-fluorescence will not decrease. The addition of
PC/Chol vesicles to NBD-labeled melittin and its analogs (Fig. 5, panel A) resulted in a
sharp increase in fluorescence due to their binding to the lipid vesicles with a fast kinetics
indicating that mutations in the leucine zipper motif did not impair the membrane-binding
ability of melittin. The addition of proteinase-k (at time point 3) to melittin (profile a,
panel A, Fig. 5) in its membrane-bound state resulted in a slow decrease in NBD-
fluorescence indicating that the N-terminal of NBD-melittin to some extent protects itself
from the digestion by the proteolytic enzyme. The 50% decrease in NBD-fluorescence,
resulting from the cleavage of NBD-melittin in its membrane-bound state took place at
around 1900 sec, probably suggesting that a part of the N-terminal of melittin was inside
the lipid bilayer of zwitterionic membrane and hence not cleaved easily by proteinase-k.
Interestingly, NBD-labeled MM-1 and MM-2 were cleaved much faster when bound to
PC/Chol vesicles compared to that of NBD-melittin as evidenced by the sharp decrease in
fluorescence after the addition of proteinase-k (profiles b and c, panel A Fig. 5). The
corresponding 50% decrease in fluorescence from the membrane-bound level for NBD-
MM-1 and -MM-2 took place at ~ 120 and 40 sec respectively. The results suggest that
probably the N-termini of both the analogs were more exposed on the surface of
zwitterionic membrane than melittin with MM-2 being the most exposed one. Taken
together, the results indicate that mutations in the leucine zipper motif although did not
inhibit the binding of melittin to zwitterionic membrane, it impaired its localization onto
it. However, N-termini of melittin and its analogs were appreciably protected in
PC/PG/Chol vesicles from proteolytic cleavage as evidenced by the slow decrease in
NBD-fluorescence following the proteinase-k treatment (panel B, Fig. 5). Since very
similar results were obtained with PE/PG vesicles, profiles not presented.
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The localization of N-terminal of melittin and its analogs onto the membrane were
also detected by recording the emission maxima of their NBD-labeled versions in the
presence of lipid vesicles (panel C and D, Fig. 5). In the presence of zwitterionic
PC/Chol vesicles melittin exhibited emission maximum at 526-527 nm, which was much
shorter than 533 nm, observed for the NBD probe, located on the surface of the
membrane (43). Interestingly, NBD-labeled MM-1 and MM-2 with zwitterionic
membrane displayed fluorescence maxima at 529-530 nm (panel C, Fig. 5), which was
close to the characteristic value for the probe, located onto the membrane surface.
However, in the presence of negatively charged PC/PG/Chol lipid vesicles, NBD-labeled
melittin and both of its analogs exhibited emission maxima in the range of 525-526.5 nm
(panel D, Fig 5), indicating the location of their N-terminal slightly inside the bilayer of
this kind of membrane. Altogether, supporting the enzymatic cleavage experiments,
fluorescence emission maxima of NBD-labeled melittin and its analogs suggested that the
localization of N-terminal of melittin was affected by the mutation of leucine zipper
motif only in zwitterionic but not in negatively charged membrane.
Substitution of heptadic leucine in the leucine zipper motif impaired the assembly of
melittin in aqueous environment: In aqueous environment at high ionic strength with
increase in concentration, melittin assembles in a tetrameric helical structure as studied
by several biophysical techniques (18-20,15). We have utilized CD, fluorescence and gel
filtration techniques to look into the effect of alanine substitution in the assembly and
secondary structure of melittin in aqueous environment. With increase in concentration
in PBS containing 1.5 M NaCl, melittin adopted more and more helical structure (panel
A, Fig. 6), consistent with its helical self-association as also reported by others (18-
20,15). Contrastingly, MM-1 (panel B, Fig. 6) and MM-2 (panel C, Fig. 6) assembled
weakly as evidenced by the shape of the spectra and small increase in CD (in mean
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residue ellipticity values at 222 nm, panel D, Fig. 6) compared to that of melittin. At any
concentration the helical structures displayed by the designed analogs MM-1 and MM-2
were significantly less than that of melittin. For example, at ~ 50 µM while melittin
exhibited a α-helix content of ~ 75%, MM-1 and MM-2 displayed helix contents of ~15
and ~8% respectively. Very similar results were obtained from concentration-dependent
CD experiments with melittin and its analogs in PBS containing 0.6 M NaCl (Figure not
shown). The results (panels A-D, Fig. 6) clearly suggest that substitution of heptadic
leucine by alanine impaired the helical structure and assembly of melittin in aqueous
environment. Tryptophan fluorescence of each of the peptides was also monitored with
increase in concentration at similar experimental conditions. It was observed that for a
particular concentration, melittin exhibited emission maximum at much shorter
wavelength (~341 nm) compared to that of MM-1 (~352 nm) and MM-2 (~353 nm)
(panel E, Fig. 6). The results indicate that with increase in concentration, tryptophan of
only melittin but not of its analogs moved to a more hydrophobic environment probably
due to its self-association (18), consistent with CD studies.
Mutation in the leucine zipper motif disrupted the oligomerization of melittin in
aqueous environment: In order to get a quantitative picture about the assembly of
melittin at higher concentration and ionic strength after the substitution of heptadic
leucine by alanine, gel filtration of melittin and its analogs were performed using PBS
containing 1.5 M NaCl as the eluent. Fig. 7 shows the plot of elution volumes of known
molecular weight markers and melittin and its analogs after passing through a Sephadex
G-50 column in PBS with 1.5 M NaCl. Melittin eluted (shown in the inset) very close to
cytochrome c (mol. wt., 12,588), indicating a molecular mass, which fairly matched with
its tetrameric molecular weight (11,384), supporting the previous observation (18). There
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was a minor peak, which corresponded to its monomer peak. However, under the same
experimental conditions, both MM-1 and MM-2 eluted at higher volumes (inset Fig. 7),
slightly after aprotinin (mol. wt., 6,500), indicating the presence of their dimeric masses.
Also both MM-1 and MM-2 exhibited prominent peaks of their monomeric masses.
Taking the gel filtration, and the previous CD and fluorescence experiments together, the
results suggest that the tetrameric helical assembly of melittin at higher concentration and
high ionic strength was disturbed as a result of substitution of heptadic leucine by
alanine.
Discussion:
The results presented here indicate the identification of a leucine zipper motif,
which plays a crucial role in the hemolytic activity of melittin. To our knowledge, this is
the first report on the identification and characterization of a leucine zipper motif in this
class of naturally occurring peptides. As a measure of toxic activity, hemolytic activity
of melittin and its analogs against human red blood cells (hRBCs) were assayed. The
data clearly demonstrated that the designed analogs were much less active than the wild
type melittin in lysing the hRBCs (Fig. 2). The synergistic effect of replacement of
heptadic leucine by alanine was evident by the fact that the double alanine mutant (MM-
2) was significantly more inactive than the single alanine mutant (MM-1). However, in
contrast to the hemolytic activity, interestingly, both MM-1 and MM-2 exhibited
comparable antibacterial activity to melittin against the tested Gram positive and negative
bacteria (Table-1).
In order to understand the molecular basis of reduction in hemolytic activity and
retention of antibacterial activity of the melittin analogs and to evaluate the structural and
functional changes associated with the mutation in this motif, membrane permeability,
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secondary structure, localization onto the membrane with different lipid composition and
self-association in aqueous environment were studied with melittin, MM-1 and MM-2.
Considering the lipid component and surface charge of outer membrane of RBC and
bacterial membrane, experiments were performed with zwitterionic, PC/Chol and
negatively charged, PC/PG/Chol and PE/PG lipid vesicles. Often the results obtained
from the structural and functional studies of membrane-active peptides with these kinds
of membrane correlate with their hemolytic and antibacterial activities (10,44).
Replacement of heptadic leucine by alanine resulted in an appreciable reduction
in membrane permeability of melittin in PC/Chol vesicles (Fig. 3, panel A), indicating
clearly an essential role of the identified leucine zipper motif in the pore-forming activity
of melittin in zwitterionic membrane. However, contrastingly the melittin analogs
exhibited very similar permeability as melittin in negatively charged PC/PG/Chol and
PE/PG membrane. The results further suggest that the amino acid sequence requirements
of a peptide to permeate zwitterionic and negatively charged lipid vesicles are not the
same, consistent with a recent study that a peptide derived from E. coli toxin hemolysin E
efficiently induced permeation in the negatively charged but not in zwitterionic
membrane (32).
The kinetics of membrane permeation induced by the designed melittin analogs in
zwitterionic and negatively charged membrane showed some interesting features. For
example, the kinetics of the zwitterionic PC/Chol membrane permeation induced by the
single alanine mutant, MM-1 (1.52 µM, Inset of panel A, Fig. 3), was only slightly slower
than that of melittin although its maximum level of dissipation of diffusion potential was
significantly reduced. Contrastingly, in negatively charged PC/PG/Chol membrane, the
kinetics of membrane permeation induced by the designed melittin analogs (0.76 µM,
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Leucine zipper motif in a naturally occurring antimicrobial peptide 17
Inset of panel B, Fig. 3) were slower than that of melittin although the maximum levels of
dissipation of diffusion potential induced by MM-1 and MM-2 ultimately reached close
to that of melittin in 200-300 sec.
The molecular basis of these observations is not clear. One possible explanation
is that probably melittin needs to adopt a specific assembly in membrane in order to
induce permeation in the zwitterionic membrane. Probably, MM-1, after substitution of
one heptadic leucine by alanine, only partly retained the desired assembly of melittin to
induce permeation in zwitterionic membrane. It could be that MM-1 formed pores
almost as fast as melittin in zwitterionic membrane, but either it could not form the same
number of pores as melittin or some of the pores formed by MM-1 got closed very fast.
So the maximum level of induced membrane permeation was significantly lower than
that of melittin although the kinetics was very similar. However, the effect of
substitution of two heptadic leucines was much more prominent in the maximum level of
MM-2 induced dissipation of diffusion potential and also in its kinetics (Inset of panel A,
Fig. 3).
On the other hand, it could be that the assembly of the designed melittin analogs
in PC/PG/Chol lipid vesicles at relatively lower concentration was to some extent
different from that of melittin. Alternatively, there may be some variation in the
mechanism of membrane permeation induced by the designed analogs and melittin in
negatively charged lipid vesicles at this peptide concentration. Probably, for any of these
possible reasons, the designed analogs induced membrane permeation in PC/PG/Chol
lipid vesicles at a slower rate than melittin (Inset of panel B, Fig. 3). However, perhaps,
due to the presence of the same number of positive charges and almost similar
amphipathic character, they induced very similar levels of maximum dissipation of
diffusion potential as that of melittin. It is to be mentioned that at slightly higher
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Leucine zipper motif in a naturally occurring antimicrobial peptide 18
concentration, for example at 2.28 µM, the kinetics as well as the maximum levels of
membrane permeation induced by the melittin analogs were very close to that of melittin
(profiles not shown). This result, further suggests that whatever difference exist between
the designed melittin analogs and melittin in terms of their assembly or even in the
mechanism of permeation of negatively charged membrane that is only at relatively lower
peptide concentration.
Although melittin and its analogs exhibited similar α-helix contents in negatively
charged membrane, the secondary structures exhibited by the mutants were much less
than that of melittin in zwitterionic membrane (Fig. 4). The molecular basis of these
observations is not clear, however it seems that substitution of heptadic leucine by
alanine in the leucine zipper motif affects the secondary structure of melittin in
zwitterionic membrane but not in negatively charged membrane.
Functional activity of a membrane-associated peptide may be influenced by its
localization onto the membrane. Proteolytic cleavage experiments (Fig. 5, panel A and
B) suggest that the substitution of heptadic leucine by alanine had remarkable effect on
the localization of melittin onto the zwitterionic membrane but not onto the negatively
charged membrane. The N-terminal of melittin was located slightly inside the
zwitterionic membrane, which is consistent with other studies (45,24) whereas the
mutants were exposed on its surface. Interestingly, the N-termini of both melittin and its
analogs were located inside the bilayer of negatively charged lipid vesicles. Similar
localizations were observed for NBD-labeled dermaseptin -3 and -4, which also
dissociate like melittin (46,47) therein and have been postulated to manifest their
antimicrobial activities by carpet/detergent mechanism (48). The results of the
proteolytic cleavage experiments were supported by the emission maxima of NBD-
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Leucine zipper motif in a naturally occurring antimicrobial peptide 19
labeled peptides in the presence of zwitterionic and negatively charged lipid vesicles
(Fig. 5, panel C and D). While NBD-melittin in the presence of both zwitterionic and
negatively charged lipid vesicles and NBD-labeled analogs in the presence of negatively
charged lipid vesicles exhibited emission maxima, which were much shorter than that
observed for the probe located onto the membrane-surface, NBD-labeled MM-1 and
MM-2 in zwitterionic membrane displayed maxima, which were close to the
characteristic value (43) for the probe, located on the surface of the membrane.
Tryptophan fluorescence of unlabeled melittin and the designed mutants was also
recorded in the presence of both kinds of lipid vesicles (Figure not shown). Melittin
exhibited emission maxima at 336-337 and 334-335 nm in the presence of zwitterionic
and negatively charged lipid vesicles respectively, which support previous observations
(49) and indicate that the tryptophan residue was located in the shallow interface region
of membrane bilayer as already reported (50). While MM-1 and MM-2 exhibited very
similar emission maxima to melittin in negatively charged membrane, the maxima (338-
339 nm), exhibited by them in the presence of zwitterionic vesicles probably indicate the
location of their tryptophan residues slightly more towards the hydrophilic region of the
membrane than that of melittin.
CD, tryptophan fluorescence (Fig. 6) and gel filtration studies (Fig. 7) with
melittin and its analogs clearly indicated that secondary structure and assembly of
melittin in aqueous environment was disrupted following the substitution of heptadic
leucine by alanine. While melittin adopted a tetrameric helical assembly at relatively
higher concentration and at high ionic strength, both MM-1 and MM-2 at similar
conditions exhibited only their dimeric masses (Fig. 7) with much reduced helical
structure. It is important to mention that although under the same experimental
conditions, both the designed melittin analogs exhibited their dimeric masses, the single
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Leucine zipper motif in a naturally occurring antimicrobial peptide 20
alanine mutant (MM-1) adopted appreciably more helical structure than the double
alanine mutant (MM-2) (Fig. 6, panel B-D). Furthermore, the results suggest that the
identified leucine zipper motif is probably a key structural element, which regulates the
secondary structure and self-association of melittin in aqueous environment. Also the
tetrameric crystal structure of melittin is stabilized by the side chain interaction of the
hydrophobic amino acids (13) like leucine and isoleucine, which are present in the ‘a’
and ‘d’ positions of the identified leucine zipper motif. These observations are in
agreement with studies that suggest for a crucial role of this motif in the assembly of
protein/peptides in aqueous and membrane environments (51,52,30).
A contrasting difference in membrane permeability of melittin and its analogs in
zwitterionic and negatively charged lipid vesicles probably reflect that the mechanisms of
membrane permeation of melittin in these two kinds of membranes are different from
each other, which is in agreement with the present notion that melittin forms pore via a
barrel stave mechanism in zwitterionic membrane (21-24) and acts as a detergent in
negatively charged membrane (25,24). Probably the results suggest that substitution of
leucine by alanine disturbs only the mechanism of melittin-induced membrane
permeation in zwitterionic but not in negatively charged membrane.
Assembly or conformation of melittin-like membrane lytic peptides in aqueous
environments may influence their activity in membrane also since most likely these
peptides bind to the target membrane from the aqueous environment. The results
presented here probably indicate that a strong hydrophobic force of melittin is needed to
disrupt the zwitterionic lipid bilayer. Proteolytic cleavage experiments and emission
spectra of NBD-labeled analogs, which were recorded with peptide concentration as low
as ~ 10 –7 M revealed that while N-terminal of melittin could penetrate the bilayer of
zwitterionic membrane, its designed mutants were localized onto the same membrane
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Leucine zipper motif in a naturally occurring antimicrobial peptide 21
surface (Fig. 5). By disturbing the leucine zipper motif, the helical structure and self-
association and thus the hydrophobic force of melittin was abrogated, which may
contribute to the reduced activity of melittin analogs in zwitterionic membrane and also
against RBCs.
That the identified leucine zipper motif may play a crucial role in regulating the
toxic activity of melittin can explain several results in the literature, the molecular basis
of which were not clearly understood. For example, a critical look at the sequence of
melittin analog, designed by DeGrado et al (5), reveals that although it lacks appreciable
sequence homology to melittin, the identified leucine zipper motif with amino acids at ‘a’
and ‘d’ positions remained intact in it. Probably, for this, the analog (5) self-associated
(53) with helical structure and retained the hemolytic activity. The absence of proline
may add to its better self-association property and helical structure and thus to the higher
hemolytic activity than melittin. Probably, the deletion of amino acids at 6, 9, 13, 16 and
20 of melittin showed more drastic effect (6) on the hemolytic activity due to impairing
its helical assembly as these residues are located at crucial ‘a’ and ‘d’ positions of the
leucine zipper motif. Similarly, it can be explained why the deletion of N-terminal of
melittin had drastic effect on its hemolytic activity compared to its antimicrobial activity
(7,8,12).
In conclusion, the results demonstrate a mechanistic dissection of melittin’s
hemolytic and antibacterial activities. The results probably indicate the presence of
different sequence elements, which control the toxic and antibacterial activity of melittin.
The ability of melittin to efficiently permeate both the negatively charged and
zwitterionic membrane makes it a poor cell-selective molecule and renders it active
against both prokaryotic (e.g. antibacterial activity) and eukaryotic cells (e.g. hemolytic
activity). The results suggest that substitution of heptadic leucine by alanine disturbed
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Leucine zipper motif in a naturally occurring antimicrobial peptide 22
the helical assembly of melittin in aqueous environment, secondary structure, membrane
permeability and localization in zwitterionic membrane, which probably caused the
reduction in hemolytic activity of melittin. However, alanine substitution did not alter
much the amphipathic character of melittin, showed much less effect on its membrane
permeability, secondary structure and localization in negatively charged membrane and
presumably therefore did not affect its antibacterial activity.
Antimicrobial peptides are potential lead molecules for designing novel drugs.
However, one of the major issues is their selective lytic activity. The results presented
here probably suggest that cell selectivity in some antimicrobial peptides can be
introduced by modulating their secondary structures and/or assembly in aqueous
environment.
Acknowledgements: The authors express their sincere thanks to the Director, CDRI for
his support. The authors are thankful to the Head, RSIC, for assistance in recording the
ES-MS spectra and thank the Head, Biochemistry Division for allowing them to use the
lyophilizer. The authors acknowledge the assistance of Dr. G. R. Bhatt and Mr. R. K.
Purshottam in using the HPLC at the initial stage of the work. The authors are very much
thankful to Drs. B. Kundu and S. Sinha for their kind assistance in many ways. Richa
Verma and Aqeel Ahmad are acknowledged for their cooperation during the progress of
the work. N.A. and S.P.Y. acknowledge the receipt of a JRF and SRF respectively from
CSIR, India.
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Leucine zipper motif in a naturally occurring antimicrobial peptide 23
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Leucine zipper motif in a naturally occurring antimicrobial peptide 26
Figure Legends: Fig. 1: Panel A, designations and sequences of melittin and its analogs used in this study.
Heptadic amino acids are marked in bold, whereas mutated amino acids are marked as
bold and underlined. Panel B, helical wheel projections of the first 21 amino acids of
melittin and MM-2 with the first amino acid glycine placed in the ‘c’ position of the
heptad.
Fig. 2: Effect of mutation in the leucine zipper motif of melittin on its hemolytic activity.
Dose-dependent hemolytic activity of melittin (a), MM-1 (b) and MM-2 (c) against
human red blood cells.
Fig. 3: Membrane permeability induced by melittin and its analogs as plotted by % of
fluorescence recovery induced by the individual peptides. Panel A, plots of fluorescence
recovery induced by different concentrations of individual peptides in zwitterionic
PC/Chol lipid vesicles (68 µM, final concentration). Symbols- closed squares, melittin;
closed circles, MM-1 and closed triangles, MM-2. Inset depicts the representative
experimental profiles for melittin and its analogs at 1.52 µM as marked. Panel B, Plots of
fluorescence recovery induced by melittin, MM-1 and MM-2 at different concentrations
(Symbols are the same as panel A) in negatively charged PC/PG/Chol lipid vesicles (68
µΜ, final concentration). Inset depicts the representative experimental profiles for
melittin and its analogs as marked at 0.76 µΜ. Fluorescence recovery was measured at ~
300 sec after addition of peptides to lipid vesicles.
Fig. 4: Determination of secondary structures of melittin (15.2 µM), MM-1 (14.9 µM)
and MM-2 (14.5 µM) in PBS (panel A), in the presence of 482 µM PC/PG/Chol (panel
B) and 362 µM PC/Chol (panel C). Symbols-solid line, melittin; dashed line, MM-1 and
dotted line, MM-2.
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Leucine zipper motif in a naturally occurring antimicrobial peptide 27
Fig. 5: Determination of localization of melittin and its analogs. Proteolytic digestion of
NBD-labeled melittin (a), MM-1 (b) and MM-2 (c) in their membrane-bound states.
Panels A and B depict the experimental profiles with PC/Chol and PC/PG/Chol lipid
vesicles respectively. 1, 2 and 3 indicate the addition of NBD-labeled individual peptides
(0.20 µΜ), lipid vesicles (120 µΜ) and proteinase-k (12 µg/ml, final concentration).
Recording of fluorescence emission spectra of NBD-labeled melittin and its analogs in
the presence of PC/Chol (panel C) and PC/PG/Chol (panel D). Peptide and lipid
concentrations were the same as the proteolytic cleavage experiments. Symbols-solid
line, melittin; short dashed line, MM-1 and short dash dotted line, MM-2. Peaks of of the
spectra in panels C and D are marked by arrows.
Fig. 6: Detection of self-association of melittin and its analogs by concentration-
dependent CD and fluorescence experiments. Panels A, B and C are the plots of CD
(mdeg, millidegree) for different concentrations of melittin, MM-1 and MM-2
respectively in PBS (pH 7.4) containing 1.5 M NaCl. Panel D, the corresponding plots of
mean residue ellipticity values at 222 nm ([θ]) vs. peptide concentration (µΜ) of melittin
(a), MM-1 (b) and MM-2 (c). Panel E, fluorescence spectra of melittin and its analogs as
marked in the figure at ~18 µΜ in PBS with 1.5 M NaCl. Excitation wavelength was set
at 280 nm.
Fig. 7: Effect of alanine substitution in the leucine zipper motif of melittin on its
oligomeric state in aqueous environment as detected by gel filtration experiments. The
plot of molecular weight of myoglobin (a), cytochrome-c (b), aprotinin (c) and insulin B
chain (d) with respect to their elution volumes after passing through a sephadex G-50
(Sigma) column (diam. 1.4 cm, length 38 cm) with PBS containing 1.5 M NaCl on a
AKTA FPLC (Amersham Pharmacia Biotech) system. The elution volumes of melittin,
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Leucine zipper motif in a naturally occurring antimicrobial peptide 28
MM-1 and MM-2 are marked with (+) signs as 1,2 and 3 respectively. The flow rate was
1.0 ml/min and absorbance was recorded at 280 nm. The inset shows the gel filtration
profiles of melittin, MM-1 and MM-2 as marked. 0.2 ml of ~ 200 µM peptides were
introduced into the gel filtration column.
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Table 1. Antibacterial activity of melittin and its analogs against gram-positive and gram-negative bacteria
4.0 ± 0.20.2 ± 0.11.2 ± 0.2Tetracycline
3.6 ± 0.61.8 ± 0.24.2 ± 0.7MM-2
4.3 ± 0.402.4 ± 0.34.5 ± 0.7MM-1
3.6 ± 0.52.0 ± 0.23.9 ± 0.6Melittin
S. aureusB. subtilisE. coli DH5αPeptide/anti-biotic name
Minimal inhibitory concentration (MIC) in µM
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Figure: 1
Sequences of Melittin and its MutantsMelittin: X-NH-GIGAVLKVLTTGLPALISWIKRKRQQ-CONH2
MM-1: X-NH-GIGAVLKVLTTGAPALISWIKRKRQQ-CONH2
MM-2: X-NH-GIGAVAKVLTTGAPALISWIKRKRQQ-CONH2
X= H or NBD
'd' 'e'
'f''c'
'b''g'
'a'
ILLL L I
W G V
GVA
A T S
K P K
G T I
'd' 'e'
'f''c'
'b''g'
'a'
L L I
W G V
GVA
A T S
K P K
G T I
IAA
Melittin MM-2
A
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0 1 2 3 4 5 6 70
20
40
60
80
100
ccc
b
bbb
a
aa
a
c cb
a
Peptide (in µM)
% o
f Hem
olyt
ic A
ctiv
ity
Figure: 2
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0.0 0.5 1.0 1.5 2.0 2.50
20
40
60
80
100
0 100 200 300 4000
40
80
120
160
Time (sec)
MM-2MM-1
Melittin
Flur
esce
nce (
a.u.)
Peptide (in µM ) % o
f Flu
ores
cenc
e R
ecov
ery
A
B
0.0 0.5 1.0 1.5 2.0 2.50
20
40
60
80
100
0 100 200 300 400 5000
102030405060
Time (sec)
Flur
esce
nce (
a.u.)
MM-2MM-1
Melittin
%
of F
luor
esce
nce
Rec
over
y
Peptide ( in µM )
Figure: 3
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200 210 220 230 240 250-25
-20
-15
-10
-5
0
5
10
Wavelength (nm)[θ] x
10-3
( Deg
ree
x cm
2 x dm
ole-1
)
200 210 220 230 240 250-20
-15
-10
-5
0
5
10
[θ
] x 1
0-3( D
egre
e x
cm2 x
dmol
e-1)
Wavelength (nm)
200 210 220 230 240 250-30-25-20-15-10-50510
Wavelength (nm)[θ] x
10-3
( Deg
ree
x cm
2 x dm
ole-1
)
A B
C
Figure: 4
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500 520 540 560 580 6000
10
20
30
40
50
Wavelength (nm)
Fluo
resc
ence
(a.u
.)
C
500 520 540 560 580 6000
10
20
30
40
50
Wavelength (nm)
Fluo
resc
ence
(a.u
.)
D
0 500 1000 1500 2000 25000
20
40
60 3
21
c
b
a
Fluo
resc
ence
(a.u
.)
Time (sec)
A
0 500 1000 1500 2000 25000
20
40
60
80
cb
a
12
3
Time (sec)
Fluo
resc
ence
(a.u
.) B
Figure: 5
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300 330 360 390 420 4500
160
320
480
640
800 MM-2
MM-1
Melittin
Fl
uore
scen
ce (a
.u.)
Wavelength (nm)
0 20 40 60 80 100 120 140048
1216202428
[θ] x
10-3
( Deg
ree
x cm
2 x dm
ole-1
)Peptide concentration (in µM )
cb
a
200 210 220 230 240 250
-120
-90
-60
-30
0
30
Incr
easin
g pe
ptid
e co
nc.
C
D (m
deg)
Wavelength (nm)
200 210 220 230 240 250
-200
-160
-120
-80
-40
0
40
C
D (m
deg)
Incr
easin
g pe
ptid
e co
nc.
A
200 210 220 230 240 250-150
-120
-90
-60
-30
0
30
Incr
easin
g pe
ptid
e co
nc.
CD
(mde
g)
B
C
E
D
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Figure: 7
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Neeta Asthana, Sharada Prasad Yadav and Jimut Kanti Ghoshcrucial role in determining its hemolytic activity but not antibacterial activity
Dissection of antibacterial and toxic activity of melittin: A leucine zipper motif plays
published online October 8, 2004J. Biol. Chem.
10.1074/jbc.M408881200Access the most updated version of this article at doi:
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