Báo cáo Y học: The solution structure of gomesin, an antimicrobial cysteine-rich peptide from the spider pptx

The solution structure of gomesin, an antimicrobial cysteine-rich
peptide from the spider
Nicolas Mandard
1
, Philippe Bulet
2
, Anita Caille
1
, Sirlei Daffre
3
and Franc¸oise Vovelle
1
1
Centre de Biophysique Mole
´
culaire, CNRS, Orle
´
ans, France;
2
Institut de Biologie Mole
´
culaire et Cellulaire, CNRS, Strasbourg,
France;
3
Departamento de Parasitologia, ICB, Universidade de Sa
˜
o Paulo, Brazil
Gomesin is the first peptide isolated from s pider exhibiting
antimicrobial activities. This highly cationic peptide is
composed of 18 amino-acid residues including four cysteines
forming two disulfide linkages. The solution structure of

gomesin has been determined using proton two-dimensional
NMR (2D-NMR) and restrained molecular dynamics
calculations. The global fold of gomesin c onsists in a well-
resolved two-stranded a ntiparallel b shee t c onnected by a
noncanonical b turn. A comparison b etween the s tructures
of gomesin and protegrin-1 from porcine and androctonin
from scorpion outlines several common features in the
distribution of hydrophobic and hydrophilic residues. The
N- and C -te rmini , the b turn and one face of the b sheet are
hydrophilic, but the h ydrophobicity of the other f ace
depends on the peptide. The similarities suggest that the
molecules interact with m embranes i n a n analogous manner.
The importance of t he intramolecular d isulfide bridges in t he
biological activity of gomesin is being i nvestigated.
Keywords: spider; cysteine-rich; antimicrobial peptide;
b sheet; NMR.
In recent years, it has become widely recognized that animal
defense systems rely on inducible or constitutive expression
of antimicrobial peptides in response to bacterial and/or
fungal infe ctions. A mong these antimicrobial molecules,
open-ended cyclic cysteine-rich peptides are the most
widespread. They h ave been characterized in plants, inver-
tebrates and v ertebrates. S tructurally, t hey can be classified
into (a) peptides adopting a b sh eet structure, namely the
mammalian defensins [1]; (b) p eptides exhibiting the CSab
(cysteine stabilized a helix b sheet) motif [2] such a s defen-
sin A from Phormia terranovae [3], drosomycin from
Drosophila melanogaster [4], heliomicin from Heliothis
virescens [5], plant defensins [6]; and (c) peptides adopting
a b-hairpin-like fold, such as tachyplesins from horseshoe

crabs [7,8], porcine protegrins [9,10], thanatin from the bug
Podisus m aculiventris [11], androctonin from the scorpion
Androctonus australis [12], lactoferricin B from bovine [13]
and a 20-residue antimicrobial peptide from t he plant
Impatiens balsamina [14]. All the peptides adopting a
b hairpin structure possess a broad antimicrobial activity
spectrum. In contrast, peptides w ith a CSab motif h ave a
more restricted activity spectrum; insect defensins are
mainly active against Gram-positive bacteria whereas
drosomycin, heliomicin and plant defensins are active
exclusively against fungi.
While there are numerous reports on the structural
characterization and the three-dimensional structure of
polypeptide toxins from spider venoms (for review see [ 15]),
it is only very recently that a peptide with antimicrobial
activity has been characterized from spiders [16]. This
peptide, gomesin, is an 18-residue cysteine-rich antimicro-
bial peptide i solated from the blood cells (hemocytes) o f the
mygalomorph spider Acanthoscurria gomesiana. Gomesin
has two disulfide bridges linking Cys2 to Cys15 and Cys6 to
Cys11. In addition, gomesin c arries two post-translational
modifications: cyclization of the N-terminal glutamine into
pyroglutamic acid (pGlu or Z) and amidation of the
C-terminal arginine. The molecule is highly cationic
(pI ¼ 9.86 calculated by
EDITSEQ
from
DNA STAR
4.05
software) with t he presence of five arginines, one lysine, a

C-terminal amidation and no acidic amino acid.
Gomesin exhibits broad activity at rather l ow concentra-
tions (often below 10 l
M
) against numerous microorgan-
isms including bacteria, filamentous fungi and yeast. In
addition, this peptide was fou nd to a ffect the v iability of t he
parasite Leishmania amazonensis and to present some
hemolytic activity against human erythrocytes. Sequence
alignments suggest strong similarities with various anti-
microbial peptides adopting a b sheet structure, such as
tachyplesins, androctonin, and protegrins [16].
In this paper, we report on the elucidation of the solution
structure of gomesin using two-dimensional
1
H-NMR
spectroscopy and molecular modeling. Gomesin adopts a
well-defined b-hairpin-like structure as it co uld b e expected
from the sequence similarities with androctonin a nd prote-
grins. The structure of these three peptides are compared i n
order to d etermine th e s tructural f eatures r equired for their
biological properties.
MATERIALS AND METHODS
NMR experiments
Gomesin peptide was s ynthesized according to classical
Fmoc chemistry as described previously [16].
Correspondence to F. Vovelle, Centre de Biophysique Mole
´
culaire,
CNRS UPR 4301, Rue Charles Sadron, 45071 Orle

´
ans Cedex 2,
France. Fax: + 3 3 23863 1517, Tel.: + 33 23825 5574,
E-mail:
Abbreviations: pGlu (Z), pyroglutamic acid; PG-1, protegrin-1.
(Received 26 J uly 2001, revised 5 December 2001, accepted 2 January
2002)
Eur. J. Biochem. 269, 1190–1198 (2002) Ó FEBS 2002
The sample for NMR spectroscopy was prepared by
dissolving 4.5 mg of synthetic gomesin in 90%H
2
O/
10%D
2
O to obtain a final solution at 3.3 m
M
. The pH
was adjusted t o 3.5 with microlitre increments of HCl 1 N.
For experiments in h eavy water, 90% of the volume of th e
previous sample was lyophilized and then dissolved in
99.99% D
2
O. The remaining v olume (10%) was completed
with H
2
O to obtain a gomesin solution at 0.3 m
M
.
A conventional set of one-dimensional and two-dimen-
sional

1
H-NMR spectra in H
2
O, including DQF-COSY
[17], Clean-TOCSY [18] and NOESY [19], was acquired at
a temperature of 278 K on a VARIAN INOVA NMR
spectrometer equipped with a z-axis fi eld-gradient unit and
operating at a proton fre quency of 600 MHz. The clean-
TOCSY spe ctrum w as collected with a spin lock t ime o f
80 ms using the MLEV-17 mixing scheme [20] and
NOESY spectra were recorded with mixing times of
120 ms and 3 00 ms. Water suppression was achieved either
by presaturation for COSY and TOCSY experiments or
using the WATERGATE pulse sequence [21] for NOESY
experiments. A new series of TOCSY and NOESY spectra
in D
2
O was also recorded at 278 K. In an attempt to
overcome ambiguities in assignment due to spectral
overlap, a second set of clean-TOCSY and NOESY
spectra was performed at 285 K. Spectra were acquired
over a spectral width corresponding to 9 p .p.m. and
referenced to the residual H
2
O signal set as the c arrier
frequency (4.964 p.p.m. at 2 78 K; 4.897 p .p.m. a t 2 85 K).
All two-dimensional NMR data were processed on a
Silicon Graphics Indy O2 workstation using the
VNMR
software package (version 6.1; Varian, Inc., Palo Alto, C A,

USA). Assignments were c arried out according to classical
procedures including spin-system identification and
sequential assignment [22] on map s recorded at 278 K.
Cross-peak intensities of the NOESY map at 278 K with
the shortest mixing time 120 ms a nd recorded over 4096
data points in the F2 dimension were integrated with
XEASY
[23].
The unusual N -terminal residue (pyroglutamic acid) was
especially built for this wo rk and its coordinates and
appropriate parameters (bond length and atom charges)
were included in the libraries of
DYANA
[24,25] and
XPLOR
[26] for molecular modeling.
Structure calculations
NOESY cross-peak in tensities were converted into upper
distance limit constraints using the
CALIBA
program [25].
The minimum distance constraint between two p rotons was
limited by their van der Waals r adi (2.0 A
˚
). Moreover, in
order t o assess possible contributions from spin diffusion
effects, some NOEs only observable on the 300-ms mixing
time NOESY map were taken into account with a 6-A
˚
upper limit constraint. Each of the two disulfide bridges

was explicitly de fined b y t hree lower/upper distance limit
restraints between the sulphur and b carbon atoms of
the two cysteines i, j involved in the linkage (1.9 A
˚
<
d(Sc
i
,Sc
j
)<2.1 A
˚
;2.5A
˚
<d(Cb
i
,Sc
j
)<3.5 A
˚
;2.5A
˚
<
d(Sc
i
,Cb
j
)<3.5 A
˚
). All these constraints were brought
together in a distance restraint file used as input to initial

steps o f molecular modeling. Several s ets of 100 structures
were generated from random-built initial models using t he
annealing procedure of the variable target function
program DYANA. During these rounds of calculations,
restraints corresponding to the stereospecific assignment of
three m ethyl p rotons proposed by
GLOMSA
were incorpor-
ated in the data set [25]. The hydrogen bonds found at each
round of calculations on a majority of structures and
corresponding to atoms involved in secondary structure
elements were also introduced as constraints. A final set of
50 structures was then generated in a fin al
DYANA
run f rom
an input file taking into account the t otal set of constraints.
Twenty out of these 50
DYANA
structures were selected on
the basis of low target function values (% 1A
˚
2
)and
subjected to e nergy minimization using Powell’s algorithm
and
CHARMM
force field parameters [27] implemented in
X
-
PLOR

3.1 software. The energy calculations were
performed with a distance dependent dielectric function
e ¼ r,a12-A
˚
cut-off distance for all nonbonded interac-
tions and a force constant of 50 kcalÆmol
)1
ÆA
˚
)2
for NOE
restraint energy terms. All calculations were carried out on
a Silicon G raphics 02 R10000 workstation and the s truc-
tures were visualized with the
SYBYL
software (TRIPOS
Inc., St Louis, MO, USA). Hydrophobic potentials were
calculated with the
MOLCAD
option [28] implemented in
SYBYL
.
PROCHECK
[29] and
PROMOTIF
[30] programs were
used for s tructural analysis.
RESULTS AND DISCUSSION
Sequence-specific assignment and secondary structure
Comparison of the one-dimensional spectra of the s amples

of gomesin at 0.3 m
M
and 3.3 m
M
in aqueous solution
clearly shows the absence of any concentration-dependent
changes in the chemical shifts or peak line widths, suggesting
the monomeric state of the peptide in our experimental
conditions. The two-dimensional
1
H-NMR spectra of
gomesin were a ssigned via standard sequential assignment
methods developed by Wu
¨
thrich [22]. The entire spin
systems of individual amino-acid residues were identified
through DQF-COSY and TOCSY experiments on the
maps at 278 K. TOCSY and NOESY maps recorded at
285 K were used to clear up ambiguities in the a ssignment
of the NH-Ha cross-peaks of Arg4 d ue to the close vicinity
of its Ha chemical shift and o f the water resonance.
Moreover, dipolar connectivities on the D
2
O N OESY
spectra enable the best-defined Ha-Ha peaks to be obtained
near the r esidual water diagonal, especially between Cys2
and C ys15, Cys6 and Cys11, Arg 4 and Thr13. The splitting
of the resonance of backbone NH and Ha protons allows
complete proton assignments for the fingerprint region
(Fig. 1 ).

1
H c hemical s hifts o f g omesin are reported i n
Table 1 and the complete pathway Ha(i) ) NH(i +1)is
shown i n Fig. 2. The NOE c onnectivity diagram exhibits
d
NN
(i,i +2)andd
aN
(i,i+ 2) N OEs between the c entral
residues (Tyr7–Arg10), suggesting the presence of a turn in
this region (Fig. 3A). Strong d
aN
(i,i +1)NOEsinseg-
ments (Cys2–Cys6 and Cys11–Cys15) a re indicative of two
extended strands of b sheet. This hypothesis is c onfirmed by
the presence of long-distance Ha(i)-Ha(j) co nnectivities
detected on D
2
O maps even if d euterium exchange studies
revealed that all amide protons were quickly exchanging
with the solvent. Figure 3B shows the number of NOEs
between t wo residue s i and j with r espect to the d ifference
|i ) j|. The enhancement o f the nu mber of NOEs observed
Ó FEBS 2002 Solution structure of gomesin (Eur. J. Biochem. 269) 1191
for 5 <|i ) j|<13 is m ainly due to connectivities between
the protons of residues Cys6 a nd Cys11 (|i ) j| ¼ 5), L eu5
and Val12 (| i ) j| ¼ 7), Arg4 a nd Thr13 (|i ) j| ¼ 9), Arg3
and Tyr14 (|i ) j| ¼ 11), Cys2 and Cys15 (|i ) j| ¼ 13).
Finally, no NOE cross-peak, indicative of an oligomeric
association in solution, could b e d etected, which is consis-

tent with the high abundance of positively charged r esidues
(five arginines and one lysine) in the primary structure of the
peptide.
Structure evaluation
The three-dimensional structure of gomesin was determined
using the standard simulated annealing p rotocol of
DYANA
AND
energy minimization with
X
-
PLOR
, as described in
Materials and methods. The final restraint file c omprised a
set o f 289 distanc e restraints including 82 intraresidual, 102
sequential, 32 medium-range (2 < |i ) j| < 5) and 73 long
range (|i ) j| ‡ 5) restraints (with an average of 16 restraints
per resid ue). Long-range limits con cern mainly residues
located in t he segments corresponding to the t wo strands o f
the b sheet (pGlu1–Tyr7; Arg10–Arg16) (data not shown).
As shown i n Table 2, the 20 selected structures are in very
good agreement with all experimental data and the standard
covalent geometry. There are no violations larger than
0.3 A
˚
and t he root-mean-square deviations (rmsd) with
respect to the standard geometry are low. Both negative van
der Waals and electrostatic energy te rms are indicative of
favorable non-bonded interaction s. Moreover, the Rama-
chandran plot exhibits nearly 91% of the (/,w) angles of all

structures in the most favored regions and additional
allowed regions according to the
PROCHECK
software
nomenclature. The structure files have been deposited at
the Protein Data Bank ( with the
accession number 1KFP.
Structure description
The overall fold of gomesin is formed by a hairpin-like
structure with a two-residue extension at the C-terminal
end. This hairpin-like structure c onsists of two antiparallel
b strands (pGlu1–Tyr7 facing Arg10–Arg16) forming a
twisted s heet and connected by a four-residue turn (Tyr7–
Arg10). A s shown in the structural statistics (Table 2) and
Fig. 1. Fingerprint region of a TOCSY spec-
trum of gomesin in 9 0%H
2
O/10%D
2
Oat
5°C, pH 3.5. The spin s ystems of the am ide
protons are designated by the amino acid one-
letter code, upper case letters. The spin system
of side chain nitrogen-bond protons is indi-
cated with the amino acid one-letter, lower
case letters.
1192 N. Mandard et al. (Eur. J. Biochem. 269) Ó FEBS 2002
by superimposition of the 20 structures (Fig. 4 ), the
structures are extremely well defined. The pairwise rmsd
on the N , Ca,C¢backbone atoms of residues 1–16 i s only

0.34 A
˚
and drops to 0.17 A
˚
when calculated in the b sheet
region. Several main structural elements contribute to a
strong stabilization of the sheet. Six regular backbone-
backbone hydrogen bonds characteristic of the b sheet
structure, NH(Arg3)–O(Tyr14), O(Arg3)–NH(Tyr14),
Table 1.
1
H chemical shifts (p.p.m.) for gomesin in aqueous solution at 278K, pH 3.5.
Residue
Chemical shifts
NH Ha Hb Others
pGlu1 8.15 4.44 2.40, 2.05 Hc 2.57, 2.57
Cys2 8.88 5.48 3.02, 2.63
Arg3 9.04 4.64 1.79, 1.69 Hc 1.54, 1.54; Hd 3.18, 3.18; NHe 7.20
Arg4 8.80 5.00 1.73, 1.58 Hc 1.42, 1.42; Hd 3.03, 3.03; NHe 7.18
Leu5 9.13 4.74 1.60, 1.60 Hc 1.51; Hd 0.81, 0.81
Cys6 9.03 5.44 2.98, 2.70
Tyr7 8.76 4.59 2.94, 2.94 Hd 7.15; He 6.78
Lys8 9.17 3.58 1.69, 1.69 Hc 0.91, 0.75; Hd 1.51, 1.51; He 2.88, 2.88; NHe 7.56
Gln9 8.53 3.94 2.21, 2.21 Hc 2.25, 2.25
Arg10 7.92 4.63 1.97, 1.85 Hc 1.61, 1.50; Hd 3.21, 3.21; NHe 7.24
Cys11 8.98 5.60 2.99, 2.48
Val12 8.92 4.35 2.00 Hc 0.86, 0.71
Thr13 8.65 4.83 3.91 Hc 1.07
Tyr14 9.17 4.80 2.94, 2.85 Hd 7.05; He 6.73
Cys15 8.97 5.16 2.86, 2.86

Arg16 8.10 4.22 1.83, 1.75 Hc 1.65, 1.65; Hd 3.18, 3.18; NHe 7.21
Gly17 8.69 3.94, 3.94
Arg18 8.41 4.27 1.84, 1.70 Hc 1.59, 1.59; Hd 3.15, 3.15; NHe 7.21
8.08.59.0

3.5
4.0
4.5
5.0
5.5





11
6
15
2
14
5
3
12
7
13
8
17
9
18
1

16
10
4
Fig. 2. A mide- a region of a 120-ms mixing
time NOESY spectrum of g omesin. For the
sake of clarity, only th e intraresidue a-amide
cross-peaks are labeled.
Ó FEBS 2002 Solution structure of gomesin (Eur. J. Biochem. 269) 1193
NH(Leu5)–O(Val12), O(Leu5)–NH(Val12) are found
between the d isulfide bridges as well as O(pGlu1)–
NH(Arg16) and NH(Tyr7)–O(Arg10) located at each
extremity of the b sheet. Two i nterstrand disulfide bridges
adopt a well-defined right-handed conformation with v
SS
,
v
1
, v
2
torsion angles close to the expected values for
favorable energy conformers (Table 2). Moreover, whatever
the model co nsidered, the average distance between the Ca
atoms of the cysteine residues is small (3.75 ± 0.10 A
˚
). This
often o ccurs when d isulfide bridges link antiparallel
b-strands [31]. The backbone of the loop (Tyr7-Lys8-
Gln9-Arg10) also exhibits a well-defined conformation.
When t he structures are best fi tted on the four backbone
residues of the turn, the local pairwise rmsd of this turn is

0.22 A
˚
. The (i,i + 3) hydrogen bond between the CO group
of Tyr7 and the NH group of Arg10 closing classical b turns
is found only o n 10 out of the 20 structures. Whatever the
nomenclature used ([32] or [33]), this turn appears to be
particularly difficult to classify as Lys8 exhibits positive /
and w angles as observed i n a le ft-handed helix and t he /, w
average values ()150°,–60°)ofGln9areveryunusual.
Owing to a lack of NOE data, the conformation of the t wo
C-terminal residues Gly17 and Arg18, which are not
included in the b sheet, i s poorly defined.
Most side chains of s trand residues adopt a well-defined
conformation due to the p resence of numerous i nterstrand
NOEs. In particular, significantly low circular variances [33]
for v
1
and v
2
angles are observed for the four cysteines, for
Tyr7, Val12, Thr13 and Tyr14 residues (CV < 0.1). Low v
1
and v
2
circular variances are also observed for long chain or
bulky resid ues such as Arg4, Leu5, and Arg10 but, in these
cases, the e xtremity of their side chain i s rather fl oppy. I n
contrast, the side chains of Arg16 and Arg18 at the
Table 2. S tructural statistics o f t he 2 0 models of go mesin. R amachandran plots were calculated with
PROCHECK

and t he energy te rms were calculated
using the
CHARMM
force field.
Restraint violations, mean number per structure (min, max)
Distance restraints > 0.3 A
˚
0.7 (0, 2)
Distance restraints > 0.2 A
˚
1.6 (1, 4)
Deviation from standard geometry, mean number per structure (min, max)
Bond lengths > 0.05 A
˚
0.3 (0, 1)
Bond angles > 10° 0.2 (0, 2)
Ramachandran Maps (%)
Most favourable regions 77.0
Additional regions 13.7
Cysteine side chain torsion angles (average values in degrees)
i ) j v
i
1
v
i
2
v
SS
v
j

2
v
j
1
Cys2-Cys15 )60.3 ± 3.1 )84.4 ± 4.5 103.6 ± 3.0 )84.7 ± 3.5 )70.7 ± 3.5
Cys6-Cys11 )65.7 ± 2.5 )96.0 ± 3.2 96.0 ± 1.4 )70.6 ± 2.7 )67.7 ± 3.4
Final energies (kcalÆmol
)1
)
E
total
)163 ± 11
E
electrostatic
)251 ± 11
E
vdw
)50.0 ± 2.5
E
NOE
13.2 ± 2.0
Average rmsd (N-Ca-C¢) Pairwise (A
˚
) Mean structure (A
˚
)
Whole 0.79 ± 0.32 0.51 ± 0.19
Hairpin 0.34 ± 0.08 0.24 ± 0.07
b sheet 0.17 ± 0.07 0.14 ± 0.05
Turn 0.22 ± 0.10 0.15 ± 0.07

A
d
NN
(i,i+1)
d
αN
(i,i+1)
d
βN
(i,i+1)
d
NN
(i,i+2)
d
αN
(i,i+2)
d
αN
(i,i+4)
5
Z CRRL CYKQ
10
RC

VT Y
15
CRGR
0 4 8 12 16
0
20

40
60
80
100
120
Range |i-j|
Number of NOEs
B
Fig. 3. N OE connectivities and number. (A) Summary of the s eq uential
NH(i) ) NH( i +1), Ha(i))NH(i +1), Hb(i))NH(i +1), and
medium range NH(i) ) NH(i +2), Ha(i))NH(i +2), Ha(i) )
NH(i + 4) connectivities i d entified for gomesin. Th e heigh t of the bars
reflects the strength of the N OE correlation as strong, medium and
weak. ( B) Number of NOEs vs. difference |i ) j|.
1194 N. Mandard et al. (Eur. J. Biochem. 269) Ó FEBS 2002
C-terminus, but also of Gln9 in the turn, display l arge
conformational variability.
Hydrophobic potentials
The distribution of hydrophobic potentials at the Connolly
surface of gomesin are presented on Fig. 5 . The lack of
definition of the extremity of several side chains does not
significantly modify the distribution of hydrophobic
potential on the surface whatever the model chosen.
Gomesin b sheet is amphipathic, its structure clearly
displays (a) an hydrophobic face formed by a large
aggregate of hydrophobic residues (Leu5, Tyr7, Val12,
and T yr14) w hich are located on the concave surface of t he
peptide; and (b) a second face showing a globally interme-
diate potential through the presence of the two apolar
disulfide bridges, the polar (Thr13) a nd the c harged (Arg4)

side chains. T wo hydrophilic regions are located at the two
spatial extremities of the molecule, at the C-te rminus with
Arg16 and Arg18, and in the turn with the presence of
Lys8, G ln9 and Arg10.
Comparison to b-hairpin-like antimicrobial peptides
with two disulfide bridges
Gomesin shares several physico-chemical properties with
most antimicrobial peptides adopting a b-hairpin-like
structure with two disulfide bridges [2]. All of them have a
molecular mass of % 2 kDa, including a rather high
percentage of basic r esidues (over 30%). In addition, their
three-dimensional structures a re stabilized by t he presence
of two internal disulfide bridges in a parallel arrangement:
Cys
1
–Cys
4
and Cys
2
–Cys
3
. Interestingly, they all have a
broad s pectrum o f activity affecting the growth of various
microorganisms as w ell as parasites. Sequence alignments
reveal high similarities between gomesin and peptides
belonging to the families of tachyplesins and polyphemusins
from horseshoe crabs [34,35], to androctonin from scorpion
[36], a nd to PG-1 from porcine leukocytes [37]. We have
compared the three-dimensional structure of gomesin to
androctonin a nd to protegrin (PG-1) w hich coordinates a re

available in the Protein Data Bank. The three-dimensional
structure o f a ndroctonin h as been determined recently in
aqueous solution ([12], PDB code 1CZ6). The structure o f
PG-1 has been studied in aqueous solution [9,10], in
(CD
3
)
2
SO [9] a s we ll as in the presence of micelles of
dodecylphosphocholine [38].
Like gomesin, PG-1 contains 18 amino acids whereas
androctonin i s s ignificantly l onger with 25 residues.
Although the spacing o f the cysteine residues differs in
gomesin, androctonin and PG-1, t he three molecules adopt
a s imilar rigid pleated b sheet structure. The two pairs of
cysteine re sidues a re separated by t hree re sidues in gomesin
instead of only one in protegrin [37]. Androctonin presents
an unequal number of residues on each s trand b etween the
two bridges, fi ve in the N-terminal strand and t hree in the
C-terminal strand. This leads to a higher t wist of the b sheet
of androctonin compared to the two other peptides. Despite
such differences, the rmsd of the coordinates of the b strands
of the three peptides when superimposed on the backbone
atoms N, Ca,C¢are very low, 0.85 A
˚
between gomesin
and androctonin and 0.87 A
˚
between gomesin and prote-
grin (1.25 A

˚
between protegrin and androctonin). On t he
basis of this best-fit superposition, we were able to perform a
structural alignment of the three molecules (Fig. 6) which
differs slightly from the sequence alignment presented by
Silva Jr. et al. [16]. The three structures are stabilized by two
tight disulfide linkages and a regular pattern of backbone-
backbone hydroge n bonds typical of antiparallel b strands
(pGlu–Tyr7 and Arg10–Arg11 in gomesin vs. Leu5–Arg9
Fig. 4. R epresentations of the polypeptide bac kbone of go mesin and o f the central hydrophobic c luster. (A) stereoview of a superposition of the
backbones of the 20 fin al structures. T he structures are b est fitted o n the N -Ca-C¢ atoms of the well-defined b shee t. (B) schematic representation of
the overall fold with the b strands represented a s arrows.
Ó FEBS 2002 Solution structure of gomesin (Eur. J. Biochem. 269) 1195
and P he12–Val16 in PG-1; Arg5–Arg11 and Gly15–Thr21
in androctonin). As with gomesin, the b turn of PG-1 is
locally well d efined and adopts an unclassified conforma-
tion. Nevertheless, the conformations of the two turns a re
different. In the c ase o f PG-1, it seems subjected to a r igid-
group Ôhinge movementÕ relative to the b sheet [9,10] and can
adopt different orientations with respect to the rigid
remaining part of the molecule. In androctonin, the two
strands of the b shee t are not c onnected by a b turn, but
instead, the ch ain re versal is ensured by a fi ve membered -
turn locally well defined. The structures of gomesin and
androctonin are particularly w ell defined in the b sheet
region. PG-1 shows a higher flexibility in water as pointed
out by much larger rmsd (with respect to the average
structure), 1.38 A
˚
and 0.8 A

˚
for the hairpin region
in references [9,10], respectively, compared to 0.14 A
˚
for
gomesin. Nevertheless, addition of (CD3)
2
SO reduces the
flexibility of the PG-1 molecule [9].
Comparison of hydrophilic/hydrophobic properties on
the molecule surfaces shows that gomesin and PG-1
structures share two highly hydrophilic and positively
charged poles located in the N- and C-terminal regions
and i n the turn (PG-1: Arg9, Arg10, A rg11; gomesin: L ys8,
Gln9, A rg10) (Fig. 5). The turn of androctonin i nvolving
three arginines is h ighly hydrophilic and positively c harged
as well as its N-terminus (Arg1, Ser2). In contrast, t he
doublet Pro24–Tyr25 gives a hydrophobic character to the
C-terminus. A large difference concerns the d istribution of
hydrophobic/hydrophilic potentials on the surface of the
b sheet between the tails and the turn. The gomesin b sheet is
divided into two nonequivalent faces: hydrophobic s ide
chains are clustered on the concave face (Leu5, Tyr7, Val12
and Tyr14), whereas two polar side chains (Arg4, Thr13)
flanked by the apo lar disulfide bridges are located o n the
other face of gomesin. The central portion of PG-1 is
particularly hydrophobic as it contains only a polar residues
Leu5, Cys6, Tyr7, Cys8, Phe12, Cys13, Val14, Cys15 and
Val16 alternatively distributed on each side of the b sheet
[9,10]. In a ndroctonin, the highly t wisted character of t he

b sheet does n ot suggest a clear dichotomy in the d istribu-
tion of polar and apolar residues. The presence of three
charged residues (Arg5, Lys8, and Lys19) distributed on
each side of the s heet reduces considerably the hydropho-
bicity of the surface of androctonin when compared to the
two other peptides (Fig. 5 ).
Mode of action
The mode of action of the t hree peptides is not yet clearly
understood. It has been established t hat androctonin a nd
PG-1 interact with the bacterial membrane. Concerning
androctonin, biochemical e xperiments have sh own t hat the
peptide induces permeabilization of the c ytoplasmic m em-
brane and interacts with negatively charged membranes in a
monomeric form [39], suggesting a mode of action similar to
a detergent effect. O n the basis o f N MR structures, s everal
models of binding of PG-1 to the cellular membrane h ave
been proposed, some possibly with an o ligomerization of
Fig. 5. D istribution of hydrophobic potentials. Middle and right:
orthographic view of the hydrophobic potentials at the connolly
surfaces (radius 1.4 A
˚
) of gomesin (top), protegrin (middle) and
androctonin (bo ttom). Left: schematic representations of the p eptide
backbones indicating the orientation in the left o rth ographic view
pictures. Hyd rophobicity i ncreases from blue to brown while green is a
colour halfway for intermediate potentials.
Fig. 6. Structural alignment a nd schematic representation of gomesin,
protegrin- 1 a nd an dr octo nin. (A) Structural alignment of the sequences.
The alignment is obtained from the best-fitted three-dimensional
superposition of the backbone atoms. The letters in italics and bold

correspond to residues used for the best-fitted three-dimensional
superposition. The zone ingrey indicates the b sheet strand limits for the
three molecules. (B) Schematic representations of the three molecules.
1196 N. Mandard et al. (Eur. J. Biochem. 269) Ó FEBS 2002
the peptides [10]. These models are supported by recent
NMR studies of the peptide in the presence of dodecyl-
phosphocholine mice lles [38], s uggesting the formation of a
dimeric structure and possibly of h igher o rder associations.
Oriented C D studies on PG-1 are indicative of two possib le
orientations of the peptide with respect to the membrane,
depending on the peptide concentration, on the membrane
components and on the hydration conditions [40]. This two-
state model corresponds to (a) a functionally inactive
binding state, when protegrin in low concentration tends to
adsorb in the headgroup region of the membrane, leading
to a decrease of the thickness of the lipid bilayer, and then to
(b) an active state, when the peptide penetrates the
hydrocarbon core of the bilayer leading to the disruption
of the m embrane integrity, p robably through the formation
of pores [40]. The similarities between the three-dimensional
structure of g omesin and those o f PG-1 a nd androctonin
suggest that gomesin e xerts antibacterial activity by inter-
acting with the cytoplasmic bacterial membrane.
Differences in t he distribution of hydrophilic and hydro-
phobic residues at the surface of the three peptides may
indicate different modes of action on the membrane. This
may also a ccount for differences in the hemolytic activity of
the peptide. I ndeed, it has been suggested that, when
compared, peptides with a high content of hydrophobic
residues are more hemolytic [41]. In this respect, the

different levels of hemolytic activity in androctonin, gome-
sin and protegrin could be linked to the difference of
hydrophobicity of their central part.
The prerequisite for antibacterial activity is still contro-
versial. Over the last few years, a growing opinion argues
that only the maintenance of the hydrophobic-hydro philic
balance in those highly cationic peptides is the key point for
activity. This viewpoint has to be t aken with cau tion; in
some cases, as with tachyplesins, the presence of disulfide
bridges leading to the formation of a w ell-folded amphi-
pathic b sheet structure does not seem essential for activity
[42]. For other peptides, such as protegrins, disulfide bridges
would be necessary to ensure an antiparallel b sheet
conformation leading to an active peptide [43]. In addition,
protegrin analogues with particular amino-acid substitu-
tions that eliminate hydrogen bonding across the b sheet
have shown reduced activities [44]. To obtain a better
understanding of the importance of disulfide bridges and the
hydrophobic-hydrophilic balance on the antimicrobial
activity of gomesin, synthetic analogues of this peptide t hat
lack on e o r both cysteine d isulfides have been designed and
are now being testing against s everal strains of microorgan-
isms and euckaryotic cells. The first results obtained suggest
that both disulfide bridges are important for the mainten-
ance of the full biological a ctivity. Gomesin a nalogs with
only one bridge or linear g omesin remain active b ut with a
specificity towards particular microorganisms (S. Daffre,
Departmento d e P arasitologia, ICB, Universidade d e Sa
˜
o,

Paulo, Brazil, personal communication). The hydrophobic/
hydrophilic balance on the antimicrobial activity of gomesin
is also investigated.
In conclusi on, we h ave determined the three-dimensional
structure of gomesin which adopts a well-defined b sheet
structure like other open-ended c yclic peptides. Gomesin is
active at low concentration (below 10 l
M
) against a large
number of bacterial and fungal strains. The presence of
two disulfide bridges, C-terminal amidation as well as
N-terminus cyclization tends to protect gomesin from
proteolytic degradatio n. These properties, associated t o a
rapid killing o f various bacterial and fungal strains a nd to a
relatively low hemolytic activity [16], are enc ouraging for
potential applications of gomesin as a therapeutic agent.
In addition, gomesin constitutes a novel probe for further
studies of the interaction between b sheet peptides and
membranes, since most biochemical and biophysical s tudies
have been done on a helical structures. A better under-
standing of the action mode of these peptides is c rucial for
the development of a new generation of antibiotics.
ACKNOWLEDGEMENTS
The authors thank Dr C. Landon for her helpful comments. This work
was supported by C NRS , the University L ouis Pasteur o f Strasbourg.
We are indebted t o Dr J P. Briand for gomesin synthesis (UPR 9 021
CNRS, IBMC S trasbourg France).
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