Correlation of Three-dimensional Structures with the Antibacterial

47 Pages·2004·2.67 MB·English

JBC Papers in Press. Published on November 30, 2004 as Manuscript M410116200
Correlation of Three-dimensional Structures with the Antibacterial Activity of a
Group of Peptides Designed Based on a Non-toxic Bacterial Membrane Anchor
Guangshun Wang*, Yifeng Li, and Xia Li
The Structure-Fun Laboratory, Eppley Institute for Research in Cancer and Allied
Diseases, University of Nebraska Medical Center, 986805 Nebraska Medical Center,
Omaha, Nebraska 68198-6805
D
o
w
n
lo
a
d
e
d
Running title: Structure and activity of antibacterial peptides fro
m
h
ttp
://w
w
w
.jb
*To whom correspondence should be addressed: Guangshun Wang, Ph.D. c.o
rg
b/
y
g
u
Eppley Institute, Room 3018 e
s
t o
n
A
p
University of Nebraska Medical Center ril 1
4
, 2
0
1
9
986805 Nebraska Medical Center
Omaha, Nebraska 68198-6805, USA
Phone: [phone]
Fax: [phone]
E-mail: [email protected].
1
Copyright 2004 by The American Society for Biochemistry and Molecular Biology, Inc.
1The abbreviations are: CD, circular dichroism; DHPG, dihexanoyl phosphatidylglycerol;
DOPG, dioctanoyl phosphatidylglycerol; DQF-COSY, double-quantum filtered
correlated spectroscopy; DSS, 2,2-dimethyl-2-silapentane-5-sulfonate sodium salt;
HSQC, heteronuclear single-quantum correlation; IIAGlc, glucose-specific enzyme IIA
involved in phosphotransfer from phosphoenolpyruvate to glucose; MPP, membrane
perturbation potential; MIC, minimum inhibition concentration; NMR, nuclear magnetic
resonance; NOE, nuclear Overhauser effect; NOESY, nuclear Overhauser effect
spectroscopy; rmsd, root mean square deviation; ROESY, rotating-frame Overhauser
D
effect spectroscopy; SAS, solvent accessible surface; SDS, sodium dodecyl sulfate; o
w
n
lo
a
TALOS, torsion angle likelihood obtained from shift and sequence similarity; TOCSY, de
d
fro
m
total correlated spectroscopy. h
ttp
://w
w
w
.jb
c
.o
rg
b/
y
g
u
e
s
t o
n
A
p
ril 1
4
, 2
0
1
9
2
Abstract
To understand the functional differences between a non-toxic membrane anchor
corresponding to the N-terminal sequence of the Escherichia coli enzyme IIAGlc and a
toxic antimicrobial peptide aurein 1.2 of similar sequence, a series of peptides was
designed to bridge the gap between them. An alteration of a single residue of the
membrane anchor converted it into an antibacterial peptide. Circular dichroism spectra
indicate that all peptides are disordered in water but helical in micelles. Structures of the
peptides were determined in membrane-mimetic micelles by solution NMR spectroscopy.
D
o
The quality of the distance-based structures was improved by including backbone angle w
n
lo
a
d
restraints derived from a set of chemical shifts (1Ha, 15N, 13Ca, and 13Cb) from natural ed fro
m
h
abundance two-dimensional heteronuclear correlated spectroscopy. Different from the ttp
://w
w
membrane anchor, antibacterial peptides possess a broader and longer hydrophobic w.jb
c
.o
rg
surface, allowing a deeper penetration into the membrane, as supported by intermolecular b/
y
g
u
e
nuclear Overhauser effect (NOE) cross peaks between the peptide and short-chain st o
n
A
p
dioctanoyl phosphatidylglycerol. An attempt was made to correlate the NMR structures ril 1
4
, 2
0
of these peptides with their antibacterial activity. The activity of this group of peptides 1
9
does not correlate exactly with helicity, amphipathicity, charge, the number of charges,
the size of the hydrophobic surface, nor hydrophobic transfer free energy. However, a
correlation is established between the peptide activity and membrane-perturbation
potential, which is defined by interfacial hydrophobic patches and basic residues in the
case of cationic peptides. Indeed, 31P solid-state NMR spectroscopy of lipid bilayers
showed that the extent of lipid vesicle disruption by these peptides is proportional to their
membrane perturbation potential.
3
Recent interest in the search for alternative therapeutics is growing due to the
drug resistance problem with traditional antibiotics. Antimicrobial peptides have attracted
much attention because of their favorable properties, such as rapid killing, wide
spectrum, and rare development of drug resistance. It is believed that these properties of
antimicrobial peptides can be attributed to their ability to target bacterial membranes (1-
4).
Membrane targeting also plays a fundamental role in virus infection, intra- and
inter-cell signal transduction. For example, enzyme IIAGlc 1 from E. coli is identified as
D
o
an amphitropic protein, which can exist either in cytoplasm or by attaching to the w
n
lo
a
d
e
cytoplasmic membrane (5). Both states are essential for the protein cascade to ensure a d
fro
m
h
successful phosphoryl transfer from the high-energy molecule phosphoenolpyruvate to ttp
://w
w
the incoming glucose. Membrane association of IIAGlc is achieved through an N-terminal w
.jb
c
.o
rg
membrane anchor (hereinafter referred to as peptide A1), which, according to 2D NMR b/
y
g
u
e
characterization, forms a short three-turn amphipathic helical structure in phospholipids st o
n
A
p
(6). Because another membrane-targeting sequence similar to this anchor is conserved in ril 1
4
, 2
other species (7), the structure of the N-terminal domain of IIAGlc is a useful model for 01
9
understanding those membrane anchors (6).
To provide an analytical tool for structural and functional studies of antimicrobial
peptides, we have created a user-friendly antimicrobial peptide database
(http://aps.unmc.edu/AP/main.html) (8). It is of outstanding interest to note that the N-
terminal sequence of the above bacterial membrane anchor, GLFD, is identical to those
of 13 antibacterial peptides collected in the database. Since all these peptides start with
the sequence GLFD, we may refer to them as the GLFD family. Such a sequence
4
similarity between an E. coli membrane anchor (6) and a group of antimicrobial peptides
from Australian frogs (9) suggest that they might have originated from the same ancestral
gene a long time ago. Because both types of peptides act on bacterial membranes, one
toxic and the other nontoxic, we are curious to learn what determines their functional
differences. Therefore, we have designed a series of peptides (peptides A2, A3, and A4)
starting from the bacterial membrane anchor (peptide A1) (6) and ending at a known
antibacterial peptide, aurein 1.2 (peptide A5) (9). The sequences and select properties of
these peptides are provided in Table 1.
D
o
To understand the structure and activity relationship of this group of peptides, it is w
n
lo
a
d
e
essential to elucidate their three-dimensional structures at high resolution. Antimicrobial d
fro
m
h
peptides are usually cationic with less than 50 residues (1-4,8). Therefore, they are very ttp
://w
w
suitable for NMR studies. In the antimicrobial peptide database (8), 68 peptides (13%) w
.jb
c
.o
rg
have been investigated by traditional 2D homonuclear NMR spectroscopy in lipid- b/
y
g
u
e
mimetic environments such as organic solvents or detergent micelles (9-11). In micelles, st o
n
A
p
structures of antimicrobial peptides are usually determined based primarily on distance ril 1
4
, 2
0
restraints derived from NOEs (10-12). This is because scalar coupling data, which also 1
9
contain valuable structural information (12), are not amenable to measure by
homonuclear NMR methods as a result of line broadening of peptide signals from micelle
binding. In contrast, chemical shifts are easy to measure and provide an alternative
approach to obtaining angle restraints by using the NMR program TALOS (13).
Although TALOS-derived angle restraints have been included in the structural refinement
of isotope-labeled proteins, the use of such restraints in the structural refinement of
micelle-bound peptides without isotope labeling has not been demonstrated. We show in
5
this study the improvement of the structural quality of these peptides using backbone
angle restraints predicted by TALOS based on a set of heteronuclear chemical shifts. This
work benefited from the recent installation of a cool probe to the 600-MHz NMR
spectrometer, which allows rapid data collection in hours, even for natural abundance
peptides. High-quality structures allow a better correlation with the activity of these
peptides. While no good correlation was found between the peptide activity and
numerous structural parameters, the membrane perturbation potential as we defined
shows a nice correlation.
D
o
w
n
lo
a
d
e
d
Materials and methods fro
m
h
ttp
Materials. All antibacterial peptides (> 95%) investigated here are synthesized and ://w
w
w
.jb
purified by Genemed Synthesis, Inc. (San Francisco, CA). The primary sequences and c
.o
rg
b/
related information for these peptides can be found in Table 1. Deuterated SDS (> 99%) y g
u
e
s
t o
was obtained from Cambridge Isotope Laboratories, Inc. (Andover, MA). Protonated n A
p
ril 1
DOPG (> 98%), dioleoyl phosphatidylglycerol and dioleoyl phosphatidylethanolamine (> 4
, 2
0
1
9
99%) were purchased from Avanti Polar Lipids (Alabaster, AL). Chloroform was
removed from phospholipids under a stream of nitrogen gas followed by evaporation
under vacuum overnight. The lipids and SDS were used without further purification.
Lipid vesicles. The lipid vesicles (4 mM) of dioleoyl phosphatidylglycerol (25%) and
dioleoyl phosphatidylethanolamine (75%) (molar ratio) in 25 mM Tris buffer in D O, pD
2
7, were made as described (51) by repeated cycles of vortexing on a mixer and 30-second
heating in a water bath at 57oC.
6
Antibacterial assay. The E. coli strain K12 3000 is a gift from Dr. Alan Peterkofsky
(NIH). The antibacterial activity of the peptides was analyzed using the standard
approach of microdilution (14). In brief, a small culture was grown overnight. A fresh
culture was inoculated with a small aliquot of the overnight culture and incubated at 37oC
until the optical density reached the logarithms stage. The culture was then diluted and
partitioned into a 96-well plate with ~106 cells per well (90 ml each). The cells were then
treated with 10 ul of the peptide at a series of concentrations, allowing the minimum
D
inhibition concentration (MIC) measurement for each. The plate was then further o
w
n
lo
a
d
incubated at the same temperature overnight (~16 hours) and read on an Ultra Microplate e
d
fro
m
Reader at 620 nm (Bio-TEK Instruments). h
ttp
://w
w
w
.jb
c
CD spectroscopy. CD spectra were collected at 25oC on a Jasco J-810 spectropolarimeter .o
rg
b/
y
from 185 to 250 nm using a 0.1-mm path length cell, with a scan rate of 100 nm/min, a gu
e
s
t o
n
time constant 1.0 s, a bandwidth of 1 nm, and a sensitivity of 100 millidegrees. Each A
p
ril 1
4
spectrum is the average of 20 scans. After background subtraction, the spectrum was , 2
0
1
9
expressed in molar ellipticity.
NMR spectroscopy. For NMR measurements, the peptide concentration is typically ~2
mM in 0.6 ml of aqueous solution of 90% H O and 10% D O at pH 5.4. The pH of each
2 2
sample was adjusted by using microliter aliquots of HCl or NaOH solution and measured
directly in the 5-mm NMR tube with a micro-pH electrode (Wilmad-Labglass). Based on
detergent titrations, the peptide/SDS molar ratio was 1:40 and the peptide/DOPG ratio
was 1:5. All proton NMR data were collected at 25oC on a Varian INOVA 600 MHz
7
NMR spectrometer equipped with a triple-resonance cryoprobe. A set of NMR spectra
was collected for each peptide using States-TPPI (15). NOESY spectra (16) were
acquired at mixing times of 50, 100, and 150 ms for peptide/micelle complexes. For
peptides in water, NOESY spectra were collected at 200 ms. TOCSY experiments were
performed with a mixing time of 75 ms using a clean MLEV-17 pulse sequence (17-19).
ROSEY spectra (20) were collected at a mixing time of 35 ms. Typically, 2D
homonuclear spectra were collected with 512 increments (16-32 scans each) in t1 and 2K
data points in t2 time domains using a spectral width of 8510.6 Hz in both dimensions
with the 1H carrier on the water resonance. The water signal was suppressed by low Do
w
n
lo
a
power presaturation during both the relaxation delay and the mixing period in NOESY de
d
fro
m
experiments, and during relaxation delay only for TOCSY (18) and DQF-COSY (21) h
ttp
://w
experiments. w
w
.jb
c
.o
To obtain backbone 15N, 13Ca, and 13Cb chemical shifts, gradient-enhanced HSQC brg/
y
g
u
e
spectra (22), between 1H and 15N as well as between 1H and 13C, were collected at the st o
n
A
p
natural abundance on the Varian 600 MHz NMR spectrometer. The 1H, 15N, and 13C ril 1
4
, 2
0
carriers were set at 4.77, 118.27, and 36.37 ppm, respectively. Typically 30 increments 1
9
(128 scans) and 80 increments (256 scans) were collected for the 15N (spectral width
2,200 Hz) and aliphatic 13C (spectral width 12,000 Hz) dimensions, respectively.
31P NMR spectra for vesicles, consisting of a mixture of the E. coli lipids, were
recorded on a 500 MHz Varian NMR spectrometer (202.2 MHz 31P frequency) at 25oC.
The 90o pulse for 31P on this direct-detected probe is 9 ms. 160 k complex data points
were collected at a spectral width of 50 kHz and a relaxation delay of 1.5 s with proton
decoupling. The 31P chemical shift of phosphoric acid (85%) was set to 0.0 ppm.
8
All NMR data were processed on an Octane workstation (SGI) using the
NMRPipe software (23). The data points in the indirect dimension were doubled by liner
prediction (24). NMR data were apodized by a 63o shifted squared sine-bell window
function in both dimensions, zero-filled prior to Fourier transformation to yield a data
matrix of 2K ¥ 1K. Because anionic DSS interacts with cationic peptides (6), it was not
used as an internal chemical shift standard to prevent the detergent additive effect.
Instead, the proton chemical shifts of the peptide were referenced to the water signal,
which in turn was referenced to internal DSS at 0.00 ppm (25). 15N and 13C chemical
D
shifts were referenced based on the ratios recommended by IUPAC (26). NMR data were o
w
n
lo
a
d
analyzed with the program PIPP (27). e
d
fro
m
h
The peptide proton signals were assigned using the standard procedure (13) based ttp
://w
w
on 2D TOCSY, DQF-COSY, and NOESY spectra. 15N, 13Ca, and 13Cb chemical shifts of w.jb
c
.o
rg
the peptides were assigned on the basis of the known proton chemical shifts. Occasional b/
y
g
u
e
ambiguity in assigning heteronuclear chemical shifts as a result of overlap is removed by st o
n
A
p
comparison with a spectrum collected at a slightly different temperature, or by ril 1
4
, 2
0
comparison with standard chemical shifts as well as with the assignments of other similar 19
peptides investigated here.
Structure calculations. Three-dimensional structures of the peptides in SDS-d at pH 5.4
25
and 25oC were calculated based on both distance and angle restraints by using the
simulated annealing protocol (28) in the NIH version of X-PLOR (29, 30). To see the
impact of TALOS-derived backbone angles on the structural quality, a separate
calculation was performed for each peptide by omitting those angle restraints.
9
The distance restraints were obtained by classifying the NOE cross-peak volumes
into strong (1.8-2.8 Å), medium (1.8-3.8 Å), weak (1.8-5.0 Å), and very weak (1.8-6.0 Å)
ranges (31). The distance was calibrated on the basis of the typical NOE patterns in an a
helix (12). Peptide backbone restraints were obtained from the TALOS (13) analysis of a
set of heteronuclear chemical shifts, including 1Ha, 13Ca, 13Cb, and 15N. A broader range
(±20o) than predicted was allowed for each angle in the structural calculations. The side-
chain c angles were derived from a combined analysis of a short mixing time NOESY
1
and ROESY spectra (32). A list of the number of NMR distance and angle restraints used
D
for structural calculations of each peptide is given in Table 2. A covalent peptide ow
n
lo
a
d
structure with random f, y, and c angles but trans planar peptide bonds was used as a ed
fro
m
starting structure. All peptide structural templates were also amidated at the C-terminus http
://w
w
using X-PLOR (29). In total, 100 structures were calculated. An ensemble of 50 w
.jb
c
.o
structures with the lowest total energy was chosen for structural analysis. This final brg/
y
g
u
ensemble of accepted structures satisfies the following criteria: no NOE violations greater es
t o
n
A
p
than 0.50 Å, rmsd for bond deviations from ideality less than 0.01 Å, and rmsd for angle ril 1
4
, 2
deviations from ideality less than 5o. 01
9
The coordinates of peptides A2, A3, A4, and A5 in SDS micelles at a
peptide/SDS ratio of 1:40, pH 5.4 and 25oC have been deposited with the Protein Data
Bank (PDB entries: 1vm2, 1vm3, 1vm4, and 1vm5).
10