The solution structure of gomesin, an antimicrobial cysteine-rich peptide from the spider Nicolas Mandard1, Philippe Bulet2, Anita Caille1, Sirlei Daffre3and Franc¸oise Vovelle1 1 Centre
Trang 1The solution structure of gomesin, an antimicrobial cysteine-rich peptide from the spider
Nicolas Mandard1, Philippe Bulet2, Anita Caille1, Sirlei Daffre3and Franc¸oise Vovelle1
1
Centre de Biophysique Mole´culaire, CNRS, Orle´ans, France;2Institut de Biologie Mole´culaire et Cellulaire, CNRS, Strasbourg, France;3Departamento de Parasitologia, ICB, Universidade de Sa˜o Paulo, Brazil
Gomesin is the first peptide isolated from spider 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 consists in a
well-resolved two-stranded antiparallel b sheet connected by a
noncanonical b turn A comparison between the structures
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-termini, the b turn and one face of the b sheet are hydrophilic, but the hydrophobicity of the other face depends on the peptide The similarities suggest that the molecules interact with membranes in an analogous manner The importance of the intramolecular disulfide bridges in the biological activity of gomesin is being investigated 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 infections Among these antimicrobial molecules,
open-ended cyclic cysteine-rich peptides are the most
widespread They have been characterized in plants,
inver-tebrates and verinver-tebrates Structurally, they can be classified
into (a) peptides adopting a b sheet structure, namely the
mammalian defensins [1]; (b) peptides exhibiting the CSab
(cysteine stabilized a helix b sheet) motif [2] such as
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 maculiventris[11], androctonin from the scorpion
Androctonus australis[12], lactoferricin B from bovine [13]
and a 20-residue antimicrobial peptide from the plant
Impatiens balsamina [14] All the peptides adopting a
b hairpin structure possess a broad antimicrobial activity
spectrum In contrast, peptides with a CSab motif have 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 isolated from the blood cells (hemocytes) of the mygalomorph spider Acanthoscurria gomesiana Gomesin has two disulfide bridges linking Cys2 to Cys15 and Cys6 to Cys11 In addition, gomesin carries 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 (pI¼ 9.86 calculated by EDITSEQ from DNA STAR4.05 software) with the presence of five arginines, one lysine, a C-terminal amidation and no acidic amino acid
Gomesin exhibits broad activity at rather low concentra-tions (often below 10 lM) against numerous microorgan-isms including bacteria, filamentous fungi and yeast In addition, this peptide was found to affect the viability of the 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 1H-NMR spectroscopy and molecular modeling Gomesin adopts a well-defined b-hairpin-like structure as it could be expected from the sequence similarities with androctonin and prote-grins The structure of these three peptides are compared in order to determine the structural features required for their biological properties
M A T E R I A L S A N D M E T H O D S
NMR experiments Gomesin peptide was synthesized 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: + 33 23863 1517, Tel.: + 33 23825 5574,
E-mail: vovelle@cnrs-orleans.fr
Abbreviations: pGlu (Z), pyroglutamic acid; PG-1, protegrin-1.
(Received 26 July 2001, revised 5 December 2001, accepted 2 January
2002)
Trang 2The sample for NMR spectroscopy was prepared by
dissolving 4.5 mg of synthetic gomesin in 90%H2O/
10%D2O to obtain a final solution at 3.3 mM The pH
was adjusted to 3.5 with microlitre increments of HCl 1 N
For experiments in heavy water, 90% of the volume of the
previous sample was lyophilized and then dissolved in
99.99% D2O The remaining volume (10%) was completed
with H2O to obtain a gomesin solution at 0.3 mM
A conventional set of one-dimensional and
two-dimen-sional 1H-NMR spectra in H2O, 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 field-gradient unit and
operating at a proton frequency of 600 MHz The
clean-TOCSY spectrum was collected with a spin lock time of
80 ms using the MLEV-17 mixing scheme [20] and
NOESY spectra were recorded with mixing times of
120 ms and 300 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 D2O 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 H2O signal set as the carrier
frequency (4.964 p.p.m at 278 K; 4.897 p.p.m at 285 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, CA,
USA) Assignments were carried out according to classical
procedures including spin-system identification and
sequential assignment [22] on maps recorded at 278 K
Cross-peak intensities of the NOESY map at 278 K with
the shortest mixing time 120 ms and 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 work and its coordinates and
appropriate parameters (bond length and atom charges)
were included in the libraries ofDYANA[24,25] andXPLOR
[26] for molecular modeling
Structure calculations
NOESY cross-peak intensities were converted into upper
distance limit constraints using the CALIBA program [25]
The minimum distance constraint between two protons was
limited by their van der Waals radi (2.0 A˚) Moreover, in
order to 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 defined by three 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(Sci,Scj) < 2.1 A˚; 2.5 A˚ < d(Cbi,Scj) < 3.5 A˚; 2.5 A˚ <
d(Sci,Cbj) < 3.5 A˚) All these constraints were brought
together in a distance restraint file used as input to initial
steps of molecular modeling Several sets of 100 structures
were generated from random-built initial models using the
annealing procedure of the variable target function
program DYANA During these rounds of calculations, restraints corresponding to the stereospecific assignment of three methyl protons proposed byGLOMSAwere 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 finalDYANArun from
an input file taking into account the total set of constraints Twenty out of these 50DYANAstructures were selected on the basis of low target function values ( 1A˚2) and subjected to energy minimization using Powell’s algorithm and CHARMM force field parameters [27] implemented in
X-PLOR3.1 software The energy calculations were performed with a distance dependent dielectric function
e¼ r, a 12-A˚ cut-off distance for all nonbonded interac-tions and a force constant of 50 kcalÆmol)1ÆA˚)2for NOE restraint energy terms All calculations were carried out on
a Silicon Graphics 02 R10000 workstation and the struc-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] andPROMOTIF[30] programs were used for structural analysis
R E S U L T S A N D D I S C U S S I O N
Sequence-specific assignment and secondary structure Comparison of the one-dimensional spectra of the samples
of gomesin at 0.3 mM and 3.3 mM 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 1H-NMR spectra of gomesin were assigned 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 assignment
of the NH-Ha cross-peaks of Arg4 due to the close vicinity
of its Ha chemical shift and of the water resonance Moreover, dipolar connectivities on the D2O NOESY spectra enable the best-defined Ha-Ha peaks to be obtained near the residual water diagonal, especially between Cys2 and Cys15, Cys6 and Cys11, Arg4 and Thr13 The splitting
of the resonance of backbone NH and Ha protons allows complete proton assignments for the fingerprint region (Fig 1) 1H chemical shifts of gomesin are reported in Table 1 and the complete pathway Ha(i)) NH(i + 1) is shown in Fig 2 The NOE connectivity diagram exhibits
dNN(i,i + 2) and daN(i,i + 2) NOEs between the central residues (Tyr7–Arg10), suggesting the presence of a turn in this region (Fig 3A) Strong daN(i,i + 1) NOEs in seg-ments (Cys2–Cys6 and Cys11–Cys15) are indicative of two extended strands of b sheet This hypothesis is confirmed by the presence of long-distance Ha(i)-Ha(j) connectivities detected on D2O maps even if deuterium exchange studies revealed that all amide protons were quickly exchanging with the solvent Figure 3B shows the number of NOEs between two residues i and j with respect to the difference
|i) j| The enhancement of the number of NOEs observed
Trang 3for 5<|i) j|<13 is mainly due to connectivities between
the protons of residues Cys6 and Cys11 (|i) j| ¼ 5), Leu5
and Val12 (|i) j| ¼ 7), Arg4 and 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 be detected, which is
consis-tent with the high abundance of positively charged residues
(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 protocol ofDYANA
AND energy minimization with X-PLOR, as described in
Materials and methods The final restraint file comprised a
set of 289 distance 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 residue) Long-range limits concern mainly residues
located in the segments corresponding to the two strands of
the b sheet (pGlu1–Tyr7; Arg10–Arg16) (data not shown)
As shown in 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 the root-mean-square deviations (rmsd) with respect to the standard geometry are low Both negative van der Waals and electrostatic energy terms are indicative of favorable non-bonded interactions 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 (http://www.rcsb.org/pdb) 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 consists of two antiparallel
b strands (pGlu1–Tyr7 facing Arg10–Arg16) forming a twisted sheet and connected by a four-residue turn (Tyr7– Arg10) As shown in the structural statistics (Table 2) and
Fig 1 Fingerprint region of a TOCSY spec-trum of gomesin in 90%H 2 O/10%D 2 O at
5 °C, pH 3.5 The spin systems of the amide protons are designated by the amino acid one-letter code, upper case one-letters The spin system
of side chain nitrogen-bond protons is indi-cated with the amino acid one-letter, lower case letters.
Trang 4by 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 is 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
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
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
8.0 8.5
9.0
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 Amide-a region of a 120-ms mixing
time NOESY spectrum of gomesin For the
sake of clarity, only the intraresidue a-amide
cross-peaks are labeled.
Trang 5NH(Leu5)–O(Val12), O(Leu5)–NH(Val12) are found between the disulfide bridges as well as O(pGlu1)– NH(Arg16) and NH(Tyr7)–O(Arg10) located at each extremity of the b sheet Two interstrand disulfide bridges adopt a well-defined right-handed conformation with vSS,
v1, v2 torsion angles close to the expected values for favorable energy conformers (Table 2) Moreover, whatever the model considered, the average distance between the Ca atoms of the cysteine residues is small (3.75 ± 0.10 A˚) This often occurs when disulfide bridges link antiparallel b-strands [31] The backbone of the loop (Tyr7-Lys8-Gln9-Arg10) also exhibits a well-defined conformation When the structures are best fitted 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 on 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 in a left-handed helix and the /, w average values ()150°,–60°) of Gln9 are very unusual Owing to a lack of NOE data, the conformation of the two C-terminal residues Gly17 and Arg18, which are not included in the b sheet, is poorly defined
Most side chains of strand residues adopt a well-defined conformation due to the presence of numerous interstrand NOEs In particular, significantly low circular variances [33] for v1and v2angles are observed for the four cysteines, for Tyr7, Val12, Thr13 and Tyr14 residues (CV < 0.1) Low v1 and v2circular variances are also observed for long chain or bulky residues such as Arg4, Leu5, and Arg10 but, in these cases, the extremity of their side chain is rather floppy In contrast, the side chains of Arg16 and Arg18 at the
Table 2 Structural statistics of the 20 models of gomesin Ramachandran plots were calculated with PROCHECK and the energy terms 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)
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 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
5
Z CRRL CY KQ
10
RC V T Y
15
CRGR
0
20
40
60
80
100
120
Range |i-j|
B
Fig 3 NOE connectivities and number (A) Summary of the sequential
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 identified for gomesin The height of the bars
reflects the strength of the NOE correlation as strong, medium and
weak (B) Number of NOEs vs difference |i ) j|.
Trang 6C-terminus, but also of Gln9 in the turn, display large
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 Tyr14) which are located on the concave surface of the
peptide; and (b) a second face showing a globally
interme-diate potential through the presence of the two apolar
disulfide bridges, the polar (Thr13) and the charged (Arg4)
side chains Two hydrophilic regions are located at the two
spatial extremities of the molecule, at the C-terminus with
Arg16 and Arg18, and in the turn with the presence of
Lys8, Gln9 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 residues (over 30%) In addition, their
three-dimensional structures are stabilized by the presence
of two internal disulfide bridges in a parallel arrangement:
Cys1–Cys4 and Cys2–Cys3 Interestingly, they all have a
broad spectrum of activity affecting the growth of various
microorganisms as well 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], and to PG-1 from porcine leukocytes [37] We have compared the three-dimensional structure of gomesin to androctonin and to protegrin (PG-1) which coordinates are available in the Protein Data Bank The three-dimensional structure of androctonin has been determined recently in aqueous solution ([12], PDB code 1CZ6) The structure of PG-1 has been studied in aqueous solution [9,10], in (CD3)2SO [9] as well as in the presence of micelles of dodecylphosphocholine [38]
Like gomesin, PG-1 contains 18 amino acids whereas androctonin is significantly longer with 25 residues Although the spacing of the cysteine residues differs in gomesin, androctonin and PG-1, the three molecules adopt
a similar rigid pleated b sheet structure The two pairs of cysteine residues are separated by three residues in gomesin instead of only one in protegrin [37] Androctonin presents
an unequal number of residues on each strand between the two bridges, five in the N-terminal strand and three in the C-terminal strand This leads to a higher twist 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 proteprote-grin and androctonin) On the 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 hydrogen bonds typical of antiparallel b strands (pGlu–Tyr7 and Arg10–Arg11 in gomesin vs Leu5–Arg9
Fig 4 Representations of the polypeptide backbone of gomesin and of the central hydrophobic cluster (A) stereoview of a superposition of the backbones of the 20 final structures The structures are best fitted on the N-Ca-C¢ atoms of the well-defined b sheet (B) schematic representation of the overall fold with the b strands represented as arrows.
Trang 7and Phe12–Val16 in PG-1; Arg5–Arg11 and Gly15–Thr21
in androctonin) As with gomesin, the b turn of PG-1 is
locally well defined and adopts an unclassified
conforma-tion Nevertheless, the conformations of the two turns are
different In the case of PG-1, it seems subjected to a
rigid-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 sheet are not connected by a b turn, but
instead, the chain reversal is ensured by a five
membered-turn locally well defined The structures of gomesin and
androctonin are particularly well 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)2SO 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 in the turn (PG-1: Arg9, Arg10, Arg11; gomesin: Lys8,
Gln9, Arg10) (Fig 5) The turn of androctonin involving three arginines is highly hydrophilic and positively charged
as well as its N-terminus (Arg1, Ser2) In contrast, the doublet Pro24–Tyr25 gives a hydrophobic character to the C-terminus A large difference concerns the distribution 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 side chains are clustered on the concave face (Leu5, Tyr7, Val12 and Tyr14), whereas two polar side chains (Arg4, Thr13) flanked by the apolar disulfide bridges are located on the other face of gomesin The central portion of PG-1 is particularly hydrophobic as it contains only apolar residues Leu5, Cys6, Tyr7, Cys8, Phe12, Cys13, Val14, Cys15 and Val16 alternatively distributed on each side of the b sheet [9,10] In androctonin, the highly twisted character of the
b sheet does not suggest a clear dichotomy in the distribu-tion of polar and apolar residues The presence of three charged residues (Arg5, Lys8, and Lys19) distributed on each side of the sheet 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 three peptides is not yet clearly understood It has been established that androctonin and PG-1 interact with the bacterial membrane Concerning androctonin, biochemical experiments have shown that the peptide induces permeabilization of the cytoplasmic mem-brane and interacts with negatively charged memmem-branes in a monomeric form [39], suggesting a mode of action similar to
a detergent effect On the basis of NMR structures, several models of binding of PG-1 to the cellular membrane have been proposed, some possibly with an oligomerization of
Fig 5 Distribution 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 (bottom) Left: schematic representations of the peptide
backbones indicating the orientation in the left orthographic view
pictures Hydrophobicity increases from blue to brown while green is a
colour halfway for intermediate potentials.
Fig 6 Structural alignment and schematic representation of gomesin, protegrin-1 and androctonin (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 in grey indicates the b sheet strand limits for the three molecules (B) Schematic representations of the three molecules.
Trang 8the peptides [10] These models are supported by recent
NMR studies of the peptide in the presence of
dodecyl-phosphocholine micelles [38], suggesting the formation of a
dimeric structure and possibly of higher order associations
Oriented CD studies on PG-1 are indicative of two possible
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 membrane integrity, probably through the formation
of pores [40] The similarities between the three-dimensional
structure of gomesin and those of PG-1 and androctonin
suggest that gomesin exerts antibacterial activity by
inter-acting with the cytoplasmic bacterial membrane
Differences in the 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 account for differences in the hemolytic activity of
the peptide Indeed, 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-hydrophilic
balance in those highly cationic peptides is the key point for
activity This viewpoint has to be taken with caution; in
some cases, as with tachyplesins, the presence of disulfide
bridges leading to the formation of a well-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 that
lack one or both cysteine disulfides have been designed and
are now being testing against several 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 activity Gomesin analogs with
only one bridge or linear gomesin remain active but with a
specificity towards particular microorganisms (S Daffre,
Departmento de Parasitologia, ICB, Universidade de Sa˜o,
Paulo, Brazil, personal communication) The hydrophobic/
hydrophilic balance on the antimicrobial activity of gomesin
is also investigated
In conclusion, we have determined the three-dimensional
structure of gomesin which adopts a well-defined b sheet
structure like other open-ended cyclic peptides Gomesin is
active at low concentration (below 10 lM) 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 degradation These properties, associated to a rapid killing of various bacterial and fungal strains and to a relatively low hemolytic activity [16], are encouraging 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 studies have been done on a helical structures A better under-standing of the action mode of these peptides is crucial for the development of a new generation of antibiotics
A C K N O W L E D G E M E N T S
The authors thank Dr C Landon for her helpful comments This work was supported by CNRS, the University Louis Pasteur of Strasbourg.
We are indebted to Dr J.-P Briand for gomesin synthesis (UPR 9021 CNRS, IBMC Strasbourg France).
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