Báo cáo Y học: SMAP-29 has two LPS-binding sites and a central hinge docx

SMAP-29 has two LPS-binding sites and a central hinge
Brian F. Tack
1
, Monali V Sawai
1
, William R. Kearney
2
, Andrew D. Robertson
3
, Mark A. Sherman
4
,
Wei Wang
5
, Teresa Hong
5
, Lee Ming Boo
5
, Huiyuan Wu
5
, Alan J. Waring
5,6
and Robert I. Lehrer
5,7
Departments of
1
Microbiology,
2
College of Medicine NMR Facility and
3
Biochemistry, University of Iowa, IA, USA;

4
Molecular Modeling Core Facility, Beckman Research Institute of the City of Hope, Duarte, CA, USA; Departments of
5
Medicine,
6
Pediatrics and
7
Molecular Biology Institute, UCLA, CA, USA
The CD spectra of SMAP-29, a n antimicrobial peptide f rom
sheep, showed disordered structure in a queous buffers, and
significant helicity in membrane-like environments, includ-
ing SDS micelles, lipopolysaccharide (LPS) dispersions, and
trifluoroethanol buffer systems. A structure determined by
NMR in 40% perdeuterated trifluoroethanol indicated that
residues 8–17 were helical, residues 18–19 formed a hinge,
and residues 20–28 formed an ordered, hydrophobic
segment. SMAP-29 was flexible in 40% trifluoroethanol,
forming two sets of conformers that differed in the relative
orientation of the N -terminal domain. We u sed a chromo-
genic Limulus assay to determine the EC
50
of the peptide (the
concentration that bound 5 0% of the a dded LPS). Studies
with full-length and t runcated SMAP-29 molecu les revealed
that each end of the holopeptide contained an LPS-binding
domain. The higher affinity LPS-binding domain was
situated in the flexible N-terminal portion. LPS b inding to
full-length SMAP-29 showed positive cooperativity, so the
EC
50

of the peptide (2.6 l
M
) was considerably lower than
that of the individual LPS-binding domains. LPS-binding
studies with a mixture of truncated peptides revealed that
this cooperativity was primarily intramolecular (i.e. invol-
ving the N - and C-terminal LPS-binding sites of the same
peptide molecule). CAP-18
[106)142]
, an antimicrobial cath-
elicidin peptide o f rabbits, resembled SMAP-29 in that it
contained N- and C-terminal LPS-binding domains, had an
EC
50
of 2.5 l
M
, and bound LPS with positive cooperativity.
We conclude that the p resence of multiple binding sites that
function cooperatively allow peptides such as SMAP-29 and
CAP-18 to bind LPS with high affinity.
Keywords: SMAP-29; cathelicidin; LPS; binding; cooper-
ativity.
SMAP-29, an antimicrobial peptide found in sheep leuko-
cytes, possesses potent activity against a broad range of
microbial pathogens, including many Pseudomonas aeru-
ginosa strains that are highly resistant to conventional
antibiotics [1–3]. A homologous peptide, CAP-18
[106)142]
is
present in rabbit leukocytes and has received considerable

attention because of its ability t o bind LPS [4,5] and its
potent antimicrobial activity [3,6,7]. Both SMAP-29 and
CAP-18
[106)142]
are synthesized from an 18-kDa precursor
that contains a c onserved 11-kDa cathelin domain, a nd
are classified as ÔcathelicidinsÕ. The only known human
cathelicidin is hCAP-18, which carries a 37-residue, largely
a helical peptide with antimicrobial [8–10] and LPS-binding
[11] activity.
SMAP-29 permeabilizes a variety of cell membranes. It is
hemolytic for human e rythrocytes [1], renders the inner
and outer membranes of Escherichia coli permeable to
disaccharide or trisaccharide (350–600 Da) in dicator
molecules [2], and rapidly induces a massive potassium
efflux from Gram positive and Gram negative bacteria [12].
In this report, we describe the solution structure and L PS
binding properties of SMAP-29 and compare them to
rabbit and human homologues.
MATERIALS AND METHODS
Peptide synthesis and purification
Peptide synthesis reagents, in cluding Fmoc amino a cids and
coupling solvents, were obtained from PE Biosystems
(Foster C ity, CA, USA) or AnaSpec (San J ose, CA, USA).
All organic solvents used for synthesis and purification were
HPLC grade or better. The primary structures of the peptides
used in this study are found in Table 1. In general, the
peptides were made at a 0.25-mmol scale, using F astMoc
TM
chemistry on ABI 431A or 433A peptide synthesizers. We

used prederivatized polyethylene glycol-polystyrene (PEG-
PS) resins (Perseptive Biosystems, Framingham, MA, USA)
and double coupling c ycles throughout. Th e crude product
was purified by RP-HPLC on a Vydac C-18 column, using a
linear gradient of acetonitrile in dilute (0.1 or 0.085%)
trifluoroacetic acid. The molecular mass of the p roduct was
confirmed by electrospray mass spectrometry, and its purity
was confirmed by analytical HPLC a nd, in some cases, by
capillary electrophoresis.
Correspondence to R. I. Lehrer, Department of Medicine, CHS
37–062, UCLA School of Medicine, 10833 LeConte Avenue,
Los Angeles, CA 90095-1690 USA. Fax: + 1 310 206 8766,
Tel.: + 1 310 825 5340, E-mail:
Abbreviations: ATR, attenuated reflectance; CFU, colony forming
units; Dtrifluoroethanol, perdeuterated trifluoroethanol;
EC
50
, the concentration exhibiting half maximal binding;
FTIR, Fourier Transform Infrared; KDO, keto-d-octulosonic acid;
LPS, lipopolysaccharide; MEC, minimal effective concentration;
MRE, mean residue ellipticity; PGG, a synthetic ÔconsensusÕ
antimicrobial peptide.
(Received 2 0 July 200 1, revised 7 December 2001, accepted
20 December 200 1)
Eur. J. Biochem. 269, 1181–1189 (2002) Ó FEBS 2002
LPS binding
Quantitative chromogenic Limulus amoebocyte assays were
performed with a QCL-1000 kit (BioWhittaker, Walkers-
ville, MD, USA) a s previously described [11]. Briefly, the
incubations were performed in flat-bottom, nonpyrogenic

96 well tissue culture plates ( Catalog no. 3596, CostarÒ,
Cambridge, MA, USA). Stock solutions of polymyxin B
(Sigma; 7600 UÆmg
)1
), SMAP-29 and truncated variants
were prepared in endotoxin-free acidified water (0.01%
acetic acid) a nd serially diluted in this solution. First, 25 lL
of peptide solution and 25 lL of a 1-endotoxin-unit per mL
solution of E. coli 0111:B4 lipopolysaccharide were mixed
and i ncubated for 30 min at 37 °C to permit peptide–LPS
binding to reach equilibrium. Then, 50 lL o f the amoebo-
cyte lysate reagent was added and exactly 10 min later,
100 lL o f chromogenic substrate (Ac-Ile-Glu-Ala-Arg-
p-nitroanilide) was introduced. Thereafter, the i ncubation
continued at 37 °C for 20 min while liberation of p-nitro-
aniline was monito red every 60 s at 4 05 nm, with a
SpectraMax 250 Kinetic Microplate Spectrophotometer
(Molecular Devices, Sunnyvale, CA, USA). The DD
between 10 and 16 min was calculated for the control
sample (containing peptide but no LPS), a nd from this value
the DD between 10 and 1 6 min for the experimental
samples, which contained peptide plus LPS, was subtracted.
The percentage reduction in procoagulant activation was
directly proportional to the percentage of LPS bound.
Hill plots [13] were performed by plotting log
10
peptide
or lipopeptide (polymyxin B) concentrations against
log
10

[(F
I
)/(1.0 ) F
I
)], where F
I
was the fractional inhibition
of procoagulant activity observed in the chromogenic assay.
Thus, a F
I
of 0.75 would correspond to a 75% inhibition of
procoagulant activity.
Radial diffusion assays
Purified peptides were serially diluted with acidified water
(0.01% acetic acid) containing 0.1% human serum albumin
(Sigma), as described previously [11,14]. The test bacteria,
E. coli strains ML-35p, DH5a and ATCC 33780, were
grown to mid-logarithmic p hase in trypticase soy b roth and
washed. Approximately 4 · 10
5
colony forming units
(c.f.u.) per mL were incorporated into a thin (1.2 mm)
underlay gel t hat contained 1% (w/v) agarose (Sigma) in
10 m
M
sodium phosphate buffer, pH 7.4, with 0.3 mgÆmL
)1
trypticase soy broth powder and 100 m
M
NaCl. Peptides

were serially diluted in acidified water with albumin to
obtain solutions containing 250, 79.1, 25, 7.9, 2.5 or 0 .79 lg
peptideÆmL
)1
.Anarrayof3.2 mmdiameterwellswasmade
in the underlay gel, and 8-lL aliquots of the various peptide
dilutions were added to them.
After 3 h, a 10-mL overlay gel [6% (w/v) trypticase soy
broth powder, 1% agarose and 10 m
M
sodium phosphate
buffer, pH 7.4] was poured, a nd the plates were incubated
overnight to allow surviving organisms to form microcol-
onies. Zone diameters were measured to the nearest 0.1 mm
and expressed in units (1 U ¼ 0.1 mm), after first subtract-
ing the diameter of the w ell. A linear relationship existed
between the zone diameter and the log
10
of the peptide
concentration. The x-intercept of this line was determined
by a least mean squares fit, and t his value was considered to
represent the minimal effective concentration (MEC).
CD spectrometry
CD measurements were made at 25 °Cina1-mm
pathlength c ell, using a 62 DS spectropolarimeter (AVIV
Associates, Lakewood, NJ, USA), equipped w ith a thermo-
electric temperature controller. The instrument was rou-
tinely calibrated with 1 mg ÆmL
)1
(+)10-camphorsulfonic

acid [15]. Mean residue ellipticity (MRE) was expressed as
[Q]
MRE
(degÆcm
2
Ædmol
)1
). Samples contained 0.2–2.0 m
M
peptide in either 50 m
M
sodium phosphate, pH 6.0, 40%
trifluoroethanol or 0.1% (% 0.22 m
M
) lipopolysaccharide,
or 20 m
M
SDS micelles. Spectra were collected at 0.2-nm
intervals, with an averaging time of 2 s per d atum point.
The fractional helical content was estimated from t he
dichroic minimum at 222 nm, as described by Chen et al.
[16].
FTIR measurements
Infrared spectra of SMAP-29 were recorded using a Bruker
Vector 22
TM
FTIR spectrometer equipped with a deuterated
triglycine sulfate detector. Solvent spectra were obtained by
subtracting the deuterated solvent spectrum from t he
SMAP-29/trifluoroethanol buffer (40 : 60, v/v) solution.

Spectra of SMAP-29 in LPS micelles were recorded after
drying the dispersion onto a germanium attenuated reflect-
ance (ATR) crystal and hydrating the s ubstrate and sample
with D
2
O f or 2 h in a Pike horizontal ATR accessory (Pike
Technologies, Madison, WI, USA). Spectra encompassed
32 scans taken at a gain of f our and a res olution of 1 cm
)1
.
NMR
For stru cture d etermination, the 0.7-mL samples co ntained
1m
M
SMAP-29 and 50 m
M
phosphate, pH 5 .94 in either
60% D
2
O(IsotecÔ100%Õ)/40% perdeuterated trifluoroeth-
anol (Dtrifluoroethanol, Cambridge Isotope Laboratories)
or 60% H
2
O/40% Dtrifluoroethanol, and were placed in
Kontes Model 240 NMR tubes. A standard set of
DQFCOSY, TOCSY and NOESY spectra were collected
at 25 °C on a Varian 500 MHz instrument (University of
Iowa College of Medicine NMR Fac ility, IA, USA).
NOESY spectra were also collected at 10 °Cand4°C, to
assess temperature dependent changes in peptide confor-

mation. All spectra were taken with a 6000-Hz spectral
width, solvent suppression by presaturation during a 2.5-s
relaxation delay, and S tates–Haberkorn phase-sensitive
Table 1. A mino-acid sequences of peptides used in this study.
Peptide Sequence
PGG GLLRRLRKKIGEIFKKYG
SMAP-29
[1)29]
RGLRRLGRKIAHGVKKYGPTVLRIIRIAG
SMAP-29
[1)18]
RGLRRLGRKIAHGVKKYG
SMAP-29
[9)29]
KIAHGVKKYGPTVLRIIRIAG
SMAP-29
[6)25]
LGRKIAHGVKKYGPTVLRII
CAP18
[106)142]
GLRKRLRKFRNKIKEKLKKIGQKIQGLL
PKLAPRTDY
CAP18
[106)126]
GLRKRLRKFRNKIKEKLKKIG
CAP18
[109)126]
KRLRKFRNKIKEKLKKIG
CAP18
[111)126]

LRKFRNKIKEKLKKIG
1182 B. F. Tack et al. (Eur. J. Biochem. 269) Ó FEBS 2002
detection in the f-1 dimension. High power 90 ° pulse widths
were 7 ls in all spectra. DQFCOSY spectra had a
resolution of 2048 complex points in the f-2 dimension
and 600 complex points in f-1. Thirty-two transients were
averaged for each increment. The resolution of TOCSY and
NOESY spectra were 1024 complex points in f-2 and 512
complex points in f-1. In these spectra, 16 transients were
averaged per f-1 increment. TOCSY spectra were collected
with mixing times of 30, 80 and 120 ms and a spin-lock field
strength of 8000 Hz. NOESY spectra were obtained with
mixing times of 50, 100, 150 and 300 ms. Spectra were
referenced to the Dtrifluoroethanol signal at 3.88 p.p.m.
Samples for hydrogen exchange studies were prepared by
subjecting 0 .7 mL of 1 m
M
SMAP-29 solution in 50 m
M
phosphate buffer (pH 5.94) to lyophilization. To begin the
exchange study, 0.7 mL of a 6 0% D
2
O/40% Dtrifluoro-
ethanol solution was added to the lyophilate. The sample
was then immediately capped, in verted four times to m ix it,
transferred to a n NMR tube, and placed in a p re-shimmed
magnet at 25 °C. Acquisition of spectra commenced
immediately after insertion, % 90 s after mixing began.
The first spectrum was complete 5 1 s after insertion, and
new spectra were taken at 5-min intervals for 1.5 h. Each

spectrum resulted from accumulation of 16 transients
containing 4096 complex data points, with a 2 .5-s relaxation
delay and 90° pulse widths. In the exchange experiment, t he
spectral width was 6000 Hz and no p resaturation was used.
Processing of NMR data
Spectra were processed using the
VNMR
6.1
B
software
package. The apodization for DQFCOSY spectra consisted
of a 0.158-s Gaussian function, shifted by 30°,inf-2andan
unshifted 0.05-s Gaussian function in f-1. NOESY and
TOCSY spectra were apodized with a 0.079-s Gaussian
function, shifted by 30°, in f -2 dimension and by an
unshifted Gaussian of 0.04 s in the f-1 dimension. NOESY
spectra were baseline corrected prior t o peak v olume
measurements. If n eeded, a low pass digital filter was
applied on transformation to remove any remaining
solvent signal. Coupling c onstants were extracted from the
DQFCOSY spectra by modeling line shapes of aH-NH-
cross peaks. Spectra in the exchange experiments were
apodized by a 0.73-Hz Lorentz ian line broadening prior to
Fourier transformation. The spectra were then baseline-
corrected u sing a cubic s pline f unction. Amid e peak
intensities as a function of time were measured using the
VNMR
6.1
B
software. Intensity vs. time profiles for p eaks

with measurably slow exchange rates w ere then fitted to
exponential functions using
VNMR
6.1
B
.
Calculation of structures and molecular modeling
Peak volumes from the
VNMR
package were converted to
XEASY
format using the program
VNMR
2
XEASY
(W. R.
Kearney, unpublished work, available on r equest). aH-NH
coupling constants and NOESY peak volumes w ere then
used to generate geometric constraints using the
DYANA
package [17]. Examination of representative build up curves
showed no evidence of spin-diffusion, so peak volumes were
extracted from the 300 ms mixing time spectrum. In
DYANA
,
a total of 300 trial structures were created and annealed
using 170 distance constraints and eight aH-NH torsion
angle constraints derived from the NMR data. The three
hundred initial structures w ere generated with torsion angles
chosen at random. NOE constraints were added to the

standard force constant set using the
DYANA
target function
multiplier of u`
0
¼ 10 kJÆmol
)1
ÆA
˚
)2
, with all other values as
DYANA
defaults. Each structure was then annealed in 4000
steps from an initial temperature of 8000 to 0 K followed by
1000 further steps of c onjugate gradient minimization.
Table 2 details the classification of NOE distance con-
straints used to calculate the SMAP-29 solution structure.
Lipophilic potentials [18] were calculated for the lowest
energy conformer in each structural subset. The lipophilic
potential was mapped onto a Connoly style, solvent-
accessible surface generated with a 1.4-A
˚
solvent radius.
This portion of the molecular modeling was performed
using the
SYBYL
package (Tripos, Inc., St Louis, MO, USA)
on a Silicon Graphics Octane (R10000) workstation in the
College of Medicine NMR Facility at the University of
Iowa, IA, USA.

The geometry of the final refined conformer sets was
evaluated by
PROCHECK
_
NMR
[19]. The coordinates for the
40 lowest energy structures of SMAP-29 in t rifluoroethanol
buffer, together with a full list of restraints have be en
deposited in the Protein Data Ban k (accession no. 1FRY).
Modeling w as performed on a S ilicon graphics Indigo-
2R10000 High Impact workstation (Beckman Research
Institute, City of Hope Core Facility), using
INSIGHT
/
DISCOVER
97.0 software within the
DISCOVER
environment
(Molecular Simulations, San Diego, CA, USA). A ribbon
representation for the two families of SMAP-29 structures
was constructed from the simulated annealing and geometry
optimization studies for each conformer-set of coordinates
using
INSIGHT
software.
RESULTS
LPS Binding
Figure 1 shows that full length SMAP-29 bound E. coli
LPS with an apparent affinity constant (EC
50

)of2.6l
M
.
SMAP-29 [6–25], a 20-mer that lacked residues 1–5 and
26–29 of the holopeptide bound LPS weakly, with an
estimated EC
50
of % 0.7–1.0 mm. As PGG, an 18 residue
peptide ( Table 1 ) that w e used as a control, had an EC
50
of
7.1 lm, the inability of SMAP-29 [6–25] to bind LPS was
not sim ply attributable to the shorter length. Because
SMAP-29 [1–18] and SMAP-29 [9–29] had EC
50
values o f
49 l
M
and 143 l
M
, respectively, the presence of at least two
Table 2. Classification of NOE distance constraints u sed in SMAP-29
structure.
Constraint type Number of constraints
Intraresidue 64
Interresidue 106
One residue away 70
Two residues away 9
Three residues away 18
Four residues away 9

< 2.50 A
˚
42
> 2.50 and < 3.50 A
˚
58
> 3.50 A
˚
70
Ó FEBS 2002 SMAP-29: conformation and LPS binding (Eur. J. Biochem. 269) 1183
LPS binding sites was suggested, one at each end of the
molecule. The thre efold g reater affi nity of SMAP-29 [1–18]
for LPS relative to SMAP-29 [9–29] suggests that the N -
terminal binding site has higher affinity for LPS than does
the C-terminal one. The presence of two binding sites and
the sevenfold to 21-fold greater affinity of the holopeptide
for L PS, indicates that binding by the holopeptide is
cooperative. The sigmoidal shape of the SMAP-29 binding
isotherm shown in Fig. 1 also indicate s cooperativity, as
does the coefficient of 2.66 ± 0.19 when binding is graphed
on a Hill plot (Fig. 2).
To determine whether the c ooperativity was intermole-
cular or i ntramolecular, we measured LPS-binding by an
equimolar mixture of SMAP-29 [1–18] and SMAP-29 [9–29]
(Fig. 2). One of these peptides (SMAP-29 [1–18]) lacks the
C-terminal LPS-binding site while the other lacks the
N-terminal site, t herefore we anticipated that if the cooper-
ativity of SMAP-29 was primarily intermolecular ( i.e.
involving LPS binding sites on different peptide molecules),
the mixture would simulate full length SMAP-29 in its

binding. The mixture had an EC
50
of 27.8 l
M
, somewhat
better than SMAP-29 [1–18] (EC
50
of 49 l
M
), but 10-fold
higher than SMAP-29 (EC
50
of 2.6 l
M
). We therefore infer
that cooperativity in LPS-binding is primarily intramolecu-
lar, whereas b inding of one LPS-bind ing site i n a SMAP-29
molecule facilitates binding by the other site. The Hill
coefficient of t he mixture was 1.86, which, together with its
EC
50
, suggests that while some intermolecular cooperativity
exists, its contribution to overall LPS binding by SMAP-29
was relatively minor.
We also used the Limulus chromogenic assay technique to
examine LPS binding by several other peptides, including
CAP-18
[106)142]
, the 37-residue homologue of SMAP-29
in the r abbit. The results c an be seen in Fig. 3. The

CAP-18
[106)142]
binding isotherm was also s igmoidal and
hadanEC
50
of 2.6 l
M
. When these data were graphed on a
Hill plot, the coefficient was 2.78 ± 0.38 (mean ± SEM)
and the line’s correlation coefficient (r) was 0.964.
CAP-18
[106)126]
a 21-mer containing only the N-terminal
portion of the holopeptide, retained considerable affinity for
LPS (EC
50
, 12.1 l
M
), but its isotherm was not sigmoidal
and the Hill coefficient of 0.78 ± 0.065 (mean ± SE),
signified that binding events were non-cooperative.
We also studied CAP-18
[106)126]
, an 18-mer that was
identical to the 21-mer except for lacking its first three
N-terminal residues (GLR). This peptide showed a >12-fold
reduction in affinity for LPS (EC
50
¼ 151 l
M

), implicat-
ing t hese initial residues in the LPS binding site. Further
N-terminal truncation to produce a 16-mer, CAP-
18
[106)126]
, virtually abolished L PS binding, c onfirming
the existence of this N-terminal LPS binding site. In an
earlier stu dy, we examined the affinity of the human
cathelicidin, LL 3 7 for E. coli LPS. The EC
50
value was
0.36 l
M
[13] and the Hill co efficient of 2.02 was consistent
with positive c ooperativity.
Correlation between LPS binding and antimicrobial
potency
Figure 4 compares the previous EC
50
data with the minimal
effective concentrations (MECs) of PGG, SMAP-29 and
the t runcated SMAP-29 variants for three different strains
of E. coli. The two properties were correlated, as a higher
affinity for E. coli LPS was associated with a lower MEC
for the organism in the presence of approximately physio-
logical (100 m
M
) concentrations of NaCl.
CD Measurements
In aqueous phosphate buffer, SMAP-29 had a mostly

disordered conformation (Fig. 5). However, when added to
LPS dispersions or to the micelles formed by 20 m
M
SDS in
50 m
M
phosphate buffer at pH 6.0 (data not shown), a
Fig. 1. LPS binding by SMAP-29 peptides. The EC
50
values derived
from these binding isotherms were as follows: full-length SMAP-29,
2.56 l
M
;PGG,7.1l
M
; SMAP-29 [1–18], 4 9 l
M
; SMAP-29 [9–29]
,
143 l
M
; SMAP-29 [6–25], % 700 l
M
. The previously reported EC
50
values for polymyxin and LL-37 in this assay were % 30 and 360 n
M
,
respectively [11].
Fig. 2. LPS binding by a m ixture of trun cated

SMAP-29 peptides. The left panel shows
binding isotherms for S MAP-29 (d)andan
equimolar mixture of SM AP-29 [1–18] and
SMAP-29 [9–29] (s). The right panel shows a
Hill plot of these data, with the Hill
coefficients adjacent to the lines.
1184 B. F. Tack et al. (Eur. J. Biochem. 269) Ó FEBS 2002
predominantly helical conformation was evident. In 4 0%
trifluoroethanol, an organic solvent whose dielectric con-
stant r esembles those of biological membranes [20], the CD
spectrum of SMAP-29 was consistent with % 57.2%
a helical content (Fig. 5). Similar helicity was observed in
SDS (57.0% helix), and LPS (63.2% helix) micelles. At
25 °C in 40% trifluoroethanol, the apparent helicity of
0.2–2 m
M
SMAP-29 varied only from 57.0% to 59.5%,
suggesting that it d id not aggregate under these conditions.
Figure 5B shows that the CD spectra for SMAP-29 [1–18]
and SMAP-29 [9–29] in LPS micelles were indistinguishable
from each other and from SMAP-29, i ndicating that N or
C-terminal truncation of SMAP-29 did not alter its shape
drastically.
FTIR studies in trifluoroethanol buffer and LPS
The helical structure of SMAP-29 in trifluoroethanol b uffer
and LPS micelles was further characterized using FTIR. In
the trifluoroethanol buffer system SMAP-29 had a domi-
nant absorption at 1 647 cm
)1
(Fig. 5A), typical of a more

nascent helical structure [21]. When SMAP-29 was incor-
porated into LPS micelles, the helical absorption peak
shifted from 1647 cm
)1
to 1655 cm
)1
more typical of a
highly stabilized helical structure [21].
NMR structure determination in 40% trifluoroethanol
Complete sequential assignment of the two-dimensional
1
H-NMR spectra for SMAP-29 was obtained using the
Wuthrich strategy [22]. The spin systems were identified
using DQF-COSY spectra, complemented by TOCSY
spectra. P artial or complete assignments were made for all
residues e xcept Arg1. In those spin systems where complete
assignments were not possible due to spectral overlap,
backbone resonances were assigned. A total of 170 NOE
derived distance constraints w ere used t o generate t he
structure families, of which 106 were interresidue contacts
and 6 4 were intraresidue contacts. Spectral o verlap caused
rejection of a further 60 intraresidue contacts from use in the
generation of structures. The NOE contacts, angular
constraints and a-proton chemical shift index together
define the structure and are displayed in Fig. 6. No new
NH-NH or aH-NH peaks appeared in the NOESY spectra
at 10 °C to a id in the conformational analysis. Peptide
aggregation occurred at 4 °C, evidenced by significant
broadening of spectral lines. Only Lys9 and Arg26 showed
any measurable protection from NH exchange.

Of the 300 conforme rs calculated from t he NMR
constraints in Dynamics Algorithm for NMB Applica-
tions (DYANA) [17] the 192 lowest energy conformers
were retained for further structural analysis. These 192
structures partitioned into two sets, differing by a 180°
flipinthe/angle of Gly18. The results of analyzing the
structures are summarized in Tables 2 and 3. The root-
mean-square deviation (rmsd) of the backbone atoms
between residues 2 and 28 was calculated to be 2.72 A
˚
for the 98 conformers in the subset with the G ly18
/ ¼ 120° (designated the L subset). For the 94 c onformers
with the Gly18 / ¼ )60° (designated the R subset), this
global backbone atom rmsd was 2.94 A
˚
. This indicates a
high degree of conformation al disorder in th e structures.
When the sets were analyzed in segments, the presence
of high ly ordered local secondary structure became
apparent. The structure was divided into three segments:
a highly flexible region from the N-terminus through a
ÔhingeÕ at Gly7, a nearly helical s egment from Arg8
through Tyr17, and another nearly helical region follow-
ing the ÔhingeÕ at Gly18/Pro19, from Thr20 t hrough
Ala28. The backbone rmsd calculated for the whole set of
192 structures over the segment containing residues 2–6
was 1.27 A
˚
. The flexibility of this segment is apparent, as
shown b y t he wide distribution o f backbone torsion

angles for these residues (Table 3). This is also consistent
with the lack of long range inter-residue NOE contacts
along the backbone of this region (Fig. 6). From residues
8–17, the f ull set backbone rmsd is 0.72 A
˚
(Table 3), the
Fig. 3. LPS binding by CAP18 peptides. The primary sequences of
these peptides a re shown in Table 1. The EC
50
values derived from
these binding isotherms were as follows: CAP18
[106)142]
,2.5l
M
;
CAP18
[106)126]
,12.1l
M
;CAP-18
[109)126]
, 151 l
M
;CAP18
[111)126]
,
> 500 l
M
.
Fig. 4. LPS binding and antimicrobial potency. The EC

50
for binding to
E. coli 0111:B4 LPS and the minimal effective concentrations (MEC)
for E. coli appear to b e correl ated.
Ó FEBS 2002 SMAP-29: conformation and LPS binding (Eur. J. Biochem. 269) 1185
backbone torsion angle distributions are narrow, and a
large number of NOE contacts of the type expected in a
helix (Fig. 6) all indicate that this region is highly ordered
and helical. This is confirmed by the negative chemical
shift index in residues 7–12 (Fig. 6). Additional support
for the helicity of this region is the protection of Lys9 NH
from H/D exchange (k ¼ 0.0086 ± 0.0002 s
)1
), probably
indicating a hydrogen bond to Gly7 O. In the region of
residues 20–29, the backbone rmsd is 0.57 A
˚
, indicating
that this region is also highly ordered. Ha-HN contacts
are observed between Leu22 and Arg26 and between
Arg23 and Arg26. Arg26 NH displayed some protection
from H/D exchange (k ¼ 0.00124 ± 0.00005 s
)1
), proba-
bly due to a hydrogen bond with Leu22 O. The NOE
contacts, negative a-proton-chemical shift index (Fig. 6)
and NH exchange protection a re consistent with consid-
erable helicity in t his region. Representations of the
backbone and important side chains of the lowest energy
conformers of the L and R sets are presented in

Fig. 7A ,B.
Fig. 5. CD s pectra of SMAP-29. The spectra
shown in the main figure were taken in 50 m
M
phosphate buffer (d), pH 6.0; 40%
trifluoroethanol in 5 0 m
M
phosphate buffer,
pH 6.0 (s); LPS micelles in 50 m
M
phosphate
buffer,pH6.0( )Theconcentrationof
peptide was 0.2 m
M
, the t emperature was
25 °C, and t he cell path-length was 0.01 cm.
Inset (a) shows FTIR spectra of SMAP-29.
Onespectrum(ÆÆÆÆÆ) was taken in 40%
trifluoroethanol in 5 0 m
M
phosphate buffer
pH 6.0. The other (–––) was taken from a film
containing LPS and S MAP -29 at a 10 : 1
molar ratio. I nset (b) s hows CD spectra of
SMAP-29 [1–18] and SMAP-29 [9–29] in LPS
micelles in 5 0 m
M
phosphate buffer, pH 6.0.
Inset B shows the CD spectra of SMAP-29
[1–18] and SMAP-29 [9–29], t aken in LPS

micelles in 5 0 m
M
phosphate buffer, pH 6.0.
Fig. 6. Summary of the NMR constraints found for SMAP-29. (A) Distance and torsion angle constraints reflect the secondary structure of
SMAP-29. Line thickness is p ropo rtional to t he strength o f the N OE. Symbo ls represent s econdary structu re as follow s: m, helix; .,betastrand;
w, helical and b eta strand c onformations present in ensemble; d, conformations are not within helical or sheet limits. (B) T he a-proton ch emical
shift index [36] is consistent with a helical conformation in all but six residues of SMAP-29.
Table 3. R MSD for backbone atom displacements (A
˚
) and backbone angle d isplacements (°) f or global a nd segmental structural superpositions.
Residue Range RMSD(full) RMSD(L) RMSD(R) RMSD(w) RMSD(/)
2–28 3.44 2.72 2.94 29.65 39.28
2–6 1.27 1.26 1.27 60.5 51.58
8–17 0.72 0.71 0.72 18.02 30.75
20–29 0.57 0.54 0.54 26.4 33.01
1186 B. F. Tack et al. (Eur. J. Biochem. 269) Ó FEBS 2002
DISCUSSION
One aim of this study was to define the LPS-binding
domains of SMAP-29. Accordingly, we will begin by briefly
describing the structure of LPS. The conserved lipid A
domain of LPS forms most of the outer membrane bilayer
of Gram-negative bacteria, and is a mono- or di-phospho-
rylated b1 fi 6-linked glucosamine disaccharide to w hich
six or seven fatty acids are a ttached b y amide or ester bonds.
The inner core oligosaccharide region of LPS contains two
or three keto-d-octulosonic acid (KDO) molecules that are
linked to t wo heptose r esidues that may contain phosphate
or other substituents. The minimal LPS structure required
for viability in Gram negative bacteria is Re LPS, w hich
consists of lipid A, KDO and heptose residues. Re LPS is

tetra-anionic at pH 7, with two negative charges derived
from the diglucosamine phosphates and two from the KDO
carboxylate anions [23].
We found that SMAP-29, a potently a ntimicrobial
peptide of s heep leukocytes, contained two distinct binding
sites for E. coli LPS, one at each end of the molecule.
Studies with a mixture of truncated SMAP-29 peptides
indicated that these sites bound LPS cooperatively, such
that when one terminal domain bound LPS, this event
greatly facilitated binding by the other terminal domain.
By studying truncated variants of SMAP-29, we localized
the highest affinity binding site to eight N-terminal residues,
RGLRRLGR. Our NMR studies revealed that th ese
residues were in a relatively flexible region. As four of the
eight residues in this N-terminal region are arginines, this
portion of the peptide most likely binds anionic groups
present in lipid A or inner core polysaccharide regions of
LPS. Multiple arginine or lysine residues also constitute a
structural motif for L PS recognition by B actericidal/
Permeability Increasing factor (BP/I), lactoferrin, lysozyme
and the antibacterial Limulus anti-LPS factor (LALF) [24].
LPS is normally stabiliz ed by the presence of divalent
cations, and it is destabilized when these cations are
displaced by EDTA or similar agents. The apparent
dissociation constant for divalent cations bound to KDO
was reported to be 14 l
M
[25]. As the EC
50
concentrations

of SMAP-29 and rabbit C AP-18 for binding E. coli LPS
were % 2.6 l
M
, both peptides should be able to displace
divalent cations from KDO sites. As the binding of divalent
cations to inner core carboxylate anions is believed to
promote LPS packing [26], displacement of these divalent
cations by peptides could increase outer membrane dis-
order, contribute to destabilization, and facilitate peptide
insertion and penetration into the bilayer.
We identified a second LPS binding domain of SMAP-29
among its C-terminal residues, VLRIIRIA. Our NMR
studies showed this region to be ordered and helical, with
one full turn of the a helix in residues 2 2–26. Whereas the
arginines in the VLRIIRIA segment should promote
interactions with anionic moieties of LPS, the five apolar
residues may interact preferentially with LPS acyl chains
and facilitate the peptide’s i nsertion into the outer mem-
brane bilayer.
In addition to testing SMAP-29 and related peptides, we
also examined its rabbit homologue, CAP-18
[106)142].
We
found that the full length peptides, CAP-18
[106)142]
,and
ovine SMAP-29 bound E. coli 0111:B4 LPS w ith almost
identical E C
50
values, approximately 2.5 l

M
. Both peptides
had sigmoidal binding isotherm s a nd had H ill coeffi-
cients between 2 and 3, indicating positive cooperativity.
CAP-18
[106)126]
, a 21-mer lacking the 16 C-terminal residues
of the holopeptide, retained moderate affinity for LPS
(EC
50
,12.1l
M
), indicating that at least two LPS-binding
domains were present. The removal of only three N-termi-
nal residues (GLR) from the 21-mer caused a > 12-fold
reduction in affinity for LPS (EC
50
¼ 151 l
M
), and remov-
ing two additional residues (KR) all but abolished LPS
binding. C onsequently, the first five residues (GLRKR) of
CAP-18
[106)142]
form an essential part of i ts high-affinity
binding site for LPS. Their similarity to the N-terminal
residues of SMAP-29 (RGLRR) is apparent.
In earlier studies that examined binding of LL-37 to LPS,
we obtained EC
50

values of 0.36 l
M
for LL-37, along with
evidence for positive cooperativity [11]. The LPS binding
domains of LL-37 are not yet r eported, but its N-terminal
residues (LLGDFF) are different in character from those of
SMAP-29 or CAP-18. It a ppears therefore that the a helical
antimicrobial peptides of mammalian leukocytes can vary
considerably in their structural and segmental organization.
Although we did not study CAP-18 peptides by NMR, a
published report describes studies with CAP-18
[106)137]
,a
slightly truncated variant of CAP-18
[106)142]
[27]. This
peptide had a disordered structure in aqueous media, and
formed an unusually stable and r igid alpha helix in 30%
trifluoroethanol. Thus, conformational flexibility, while a
property of SMAP-29, is not a general property of such
peptides. Whether the flexible LPS-binding domain con-
tributes to the function of SMAP-29 remains to b e
determined.
Fig. 7. Models of SMAP-29 in trifluoroethanol buffer. (A) Eight superimposed dynamic models of SMAP-29, four in each conformation (A, black
backbone and B, gray backbone), calculated from simulated annealing and geometry optimization. (B) A r ibbon representation of SMAP-29 in
conformation A ( left side) and an other in conformation B ( right side). In e ach figure, the fl exible N-terminal segment is at the bottom.
Ó FEBS 2002 SMAP-29: conformation and LPS binding (Eur. J. Biochem. 269) 1187
LPS molecules normally occupy up to 80% of the outer
leaflet of t he outer membrane a nd are linked by divalent
cations to form an oriented and highly ordered structure that

provides a formidable barrier to the uptake of exogenous
peptides. Initial binding of the arginine-rich N-terminal
domains of SMAP-29 or CAP-18 to LPS molecules on the
bacterial surface is likely to involve displacement o f divalent
cations, causingincrease d mobilitya ndd isordered packingo f
the LPS molecules and t heir acyl chains. Because SMAP-29
and CAP-18 are considerably larger than the divalent cations
they replace, their insertion into t he outer leaflet would cause
the outer membrane to expand, as was recently demonstrated
for C AP-18
[106)142]
[28]. The resulting architec tural chaos
should provide the amphipathic C-terminal domain of
SMAP-29 even greater access to acyl c hains i n both leaflets
of the outer membrane.Thism echanismi s consistent with the
positive cooperativity we noted in our LPS binding studies,
and with the general f ormulations of Ôself-promoted uptakeÕ
as shown primarily by the work of Hancock [29,30].
In addition to a helical peptides such as those described
here, several other molecules that bind L PS exist, including
many with beta-sheet structures [31–33]. Recent studies
with horseshoe crab Factor C, a molecule whose autocat-
alytic activation by f emtogram concentrations of LPS
triggers hemolymph coagulation, are o f considerable
interest. Factor C is a large, multidomain protein, with a
C-terminal serine protease domain and N-terminal LPS-
binding domains. The N-terminal fragment of Factor C
bound LPS with positive cooperativity (Hill coeffi-
cient ¼ 2.2) [34], and contained three 3.5–4 kDa small
consensus repeat (S) domains. Two of these, S 1 and S3,

bound LPS with EC
50
values of 2.25 l
M
(S1) and 1 l
M
(S3), q uite similar to those of the peptides we studied [35].
Binding isotherms for the S1 fragment were sigmoidal,
and the Hill coefficient o f 2.42 indicated positive cooper-
ativity. The fragment bound to lipid A with positive
cooperativity (Hill coefficient ¼ 2.2) and contained two
LPS-binding short consensus repeat (sushi) domains, S1
and S3, each containing a 3 4-residue LPS-binding site. The
authors suggested that two factors were critical for making
Factor C acutely se nsitive to LPS; the presence of multiple
LPS-binding s ites on a single Factor C molecule and their
high positive cooperativity in LPS binding. Although the
affinity of SMAP-29 or CAP-18 for LPS we measured in
our Limulus chromogenic assays was considerably lower
than th at reported for Factor C, these p eptides a lso
contained multiple binding sites and its LPS binding
manifested positive cooperativity.
Should a Ôtake-home messageÕ be needed from the present
study, one might remember Gulliver and the Lilliputians
who shackle d him while he slept. Simply, that a multitude of
small binding events, especially when they are performed
cooperatively, can secure large objects.
ACKNOWLEDGEMENTS
This study was supported by grants from the National Institutes of
Health: AI-43934 and A I-37945 to R. I. L., and HL-61234 to P. M.

and B. T. The work was also supported, in part, by the Cystic Fibrosis
Foundation (McCray, 97 ZO; Lehrer, 97-ZO) and by intramural funds
from the University of I owa Department of Me dicine. We thank
Reviewer 2 for suggesting the binding study with a mixture of truncated
SMAP-29 peptides.
REFERENCES
1. Skerlavaj, B., Benincasa, M., Risso, A., Zanetti, M. & Gennaro, R.
(1999) SMAP-29: a potent antibacterial and antifungal peptide
from shee p leukocytes. FEBS Lett. 463, 58–62.
2. Mahoney, M.M., Lee, A.Y., Brezinski-Caliguri, D.J. & Huttner,
K.M. (1995) Molecular analysis of the sheep cathelin family
reveals a novel antimicrobial peptide. FEBS Lett. 377 , 519–522.
3. Travis, S.M., Anderson, N.N., Forsyth, W.R., Espiritu, C.,
Conway, B.D., Greenberg, E.P., McCray Jr, P.B., Lehrer, R.I.,
Welsh, M.J. & Tack, B.F. (2 000) Bactericidal activity of
mammalian cathelicidin-derived peptides. Infect. Immun. 68 ,
2748–2755.
4. Sawa, T., Kurahashi, K., Ohara, M., Gropper, M.A., Doshi, V.,
Larrick, J.W. & Wiener-Kronish, J.P. (1998) Evaluation of
antimicrobial and lipopolysaccharide-neutralizing effects of a
synthetic CAP18 fragment against Pseudomonas aeruginosa in a
mouse model. Antimicrob. A gents Chemother. 42, 3269–3275.
5. Larrick, J.W., M organ, J.G., Palings, I., Hirata, M. & Yen, M.H.
(1991) Complementary DNA sequence of rabbit CAP18 – a
unique lipopolysaccharide binding protein. Biochem. Biophys. Res.
Commun. 179, 170–175.
6. Mason, D.J., Dybowski, R., Larrick, J.W. & Gant, V.A. (1997)
Antimicrobial action of rabbit leukocyte CAP18 (106–137).
Antimicrob. Agents Chemother. 41, 624–629.
7. Tossi, A., Scocchi, M., Skerlavaj, B. & Gennaro, R. (1994)

Identification and characterization of a primary antibacterial
domain in CAP18, a lipopolysaccharide b inding protein from
rabbit leukocytes. F EBS Lett. 339, 108–112.
8. Bals, R., Weiner, D.J., Moscioni, A .D., Meegalla, R.L. & Wilson,
J.M. (1999) Augmentation of innate host defense by expression of
a cathelicidin ant imicrobial peptide. Infect. I mmun. 67, 6084–6089.
9. Johansson, J., Gudmundsson, G.H., Rottenberg, M.E., Berndt,
K.D. & Agerberth, B. (1998) Conformation-dependent antibac-
terial activity of the naturally occurring human peptide LL-37.
J. Biol. Chem. 273, 3 718–3724.
10. Agerberth, B., Gunne, H., Odeberg, J., Kogner, P., Boman, H.G.
& Gudmundsson, G.H. (1995) FALL-39, a putative human
peptide antibiotic, is cysteine-free and expressed in bone marrow
and testis. Proc. Natl Acad. Sci. USA 92, 195–199.
11. Turner, J., Cho, Y., Dinh, N.N., Waring, A.J. & Lehrer, R.I.
(1998) Activities of LL-37, a cathelin-associated antimicrobial
peptide of human neutrophils. Antimicrob. Agents Chemother. 42,
2206–2214.
12. Orlov, D., Nguyen, T. & Lehrer, R.I. (2002) Potassium release, a
useful tool for studying antimicrobial peptides. J. Microbiol.
Methods,inpress.
13. Hill, A.V. (1910) The combinations of hemoglobin wit h oxygen
andwithcarbonmonoxide.I.J. Physiol. ( London) 40,4–8.
14. Lehrer, R.I., Rosenman, M., Harwig, S.S., Jackson, R. & Eisen-
hauer, P. (1991) Ultrasensitive assays for endogenous antimicro-
bial polypeptides. J. Immunol. M ethods 137, 167–173.
15. Johnson Jr, W.C. (199 0) Protein secondary structure a nd circular
dichroism: a p ractical guide. Proteins 7, 205–214.
16. Chen, Y.H., Yang, J .T. & Chau, K.H. (1974) Determination of
the helix and beta form of protein s in a queous solution by circular

dichroism. Biochemistry 13 , 3350–3359.
17. Guntert, P., Mumenthaler, C. & Wuthrich, K. (1997) Torsion
angle dynamic s for NMR stru cture calcu lat ion with the new
program DYANA. J. Mol. Biol. 273, 2 83–298.
18. Heiden, W., Moeckel, G. & Brickmann, J. (1943) A new approach
to analysis and d isplay of local lipophilicity/hydrophilicity map-
ped on molecular surfaces. J. Comput. Aided Mol. Des. 7, 503–514.
19. Laskowski, R.A., Rullman, J.A., MacArthur, M.W., Kaptein, R.
& Thornton, J.M. (2001) AQUA and PROCHECK-NMR:
program for checking the quality of protein structures solved by
NMR. J. Biomol. NMR 8, 477–486.
1188 B. F. Tack et al. (Eur. J. Biochem. 269) Ó FEBS 2002
20. Buck, M. (1998) Trifluoroethanol and colleagues: cosolvents come
of age. Recent studies with peptides and proteins. Q. Rev. Biophys.
31, 297–355.
21. Goormaghtigh, E., Raussens, V. & Ruysschaert, J.M. (1999)
Attenuated total reflection infrared spectroscop y o f proteins
and lipids in biological membranes. Biochim. Biophys. Acta 1 422,
105–185.
22. Wuthrich, K. (1986) NMR o f Proteins and Nucleic Acids. John
Wiley & Son s, New York.
23. Din, Z .Z., Mukerjee, P., Kastowsky, M. & Takayama, K. (1993)
Effect of pH on solubility and ionic state of lipopolysaccharide
obtained from the deep rough mutant of Escherichia coli.
Biochemistry 32 , 4579–4586.
24. Ferguson, A.D., Hofmann, E., Coulto n, J.W., Diederichs, K. &
Welte, W. (1998) Siderophore-mediated iron transport: crystal
structure of Fhu A wit h b ound lip opolysaccharide. Science 282,
2215–2220.
25.Schindler,M.&Osborn,M.J.(1979)Interactionofdivalent

cations and polymyxin B with lipopolysaccharide. Biochemistry
18, 4425–4430.
26. Snyder, S., Kim, D. & McIntosh, T.J. ( 1999) Lipopo ly-
saccharide bilayer s tructure: effect of ch emotype, core mutations,
divalent cations, and te mperature. Bioc hemistry 38, 1 0758–
10767.
27. Chen, C., Brock, R., Luh, F., C hou , P.J., Larrick, J.W., Huang,
R.F. & Huang, T.H. (1995) The solution structure of the active
domain of CAP18 – a lipopolysaccharide binding protein from
rabbit leukocytes. FEBS Lett. 37 0 , 46–52.
28. Gutsmann, T., Larrick, J.W., Seydel, U. & Wiese, A. (1999)
Molecular mechanisms of interaction of rabbit CAP18 with
outer membranes of gram-negative bacteria. Biochemistry 38,
13643–13653.
29. Zhang, L., Dhillon, P., Yan, H., Farmer, S. & Hancock, R.E.
(2000) Interactions of bacterial cationic p eptide antibiotics with
outer and cytoplasmic membranes of Pseudomonas aeruginosa.
Antimicrob. A gents Chemother. 44, 3317–3321.
30. Moore, R.A. & Hancock, R.E. (1986) Involvement of outer
membrane of Pseudomonas cepacia in aminoglycoside and
polymyxin resistance. Antimicrob. Agents Chemother. 30 , 923–926.
31. Hoess, A., Watson, S., Siber, G.R. & Liddington, R. (1993)
Crystal structure of an end otoxin-neutralizing protein from the
horseshoe crab, Limulus anti-LPS factor, at 1.5 A
˚
resolution.
EMBO J. 12, 3351–3356.
32. Beamer, L.J., Carroll, S.F. & Eisenberg, D. (1998) The BPI/LBP
family of proteins: a structural analysis of conserved regions.
Protein S ci. 7, 906–914.

33. Beamer, L.J., Carroll, S.F. & Eisenberg, D. (1997) C rystal stru c-
ture of human BPI and two bound phospholipids at 2.4 angstrom
resolution. Science 276, 1861–1864.
34. Tan, N.S., Ho, B. & Ding, J.L. (2000) High-affinity LPS binding
domain(s) in recombinant f actor C of a horseshoe crab neutralizes
LPS-induced lethality. FAS EB J. 14 , 859–870.
35. Tan, N.S., Ng, M.L., Yau, Y.H., Chong, P.K., Ho, B. &
Ding, J.L. (2000) Definition of endotoxin binding sites in horse-
shoe crab factor C recombinant sushi proteins and neutralization
of endotoxin by sushi peptides. FAS EB J. 14 , 1801–1813.
36. Wishart, D .S., Sykes, B.D. & Richards, F.M. (1992) The chemical
shift index: a fast and simple m ethod for t he assignment of protein
secondary structure through NMR spectroscopy. Biochemistry 31,
1647–1651.
Ó FEBS 2002 SMAP-29: conformation and LPS binding (Eur. J. Biochem. 269) 1189