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

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

´
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

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

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

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 ).

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

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

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

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

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

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

˚
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

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

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,

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