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Acta Crystallographica Section D

Biological
Structure of the polypeptide crotamine from the
Crystallography Brazilian rattlesnake Crotalus durissus terrificus
ISSN 0907-4449

Monika A. Coronado,a,b The crystal structure of the myotoxic, cell-penetrating, basic Received 27 April 2013
Accepted 29 June 2013
Azat Gabdulkhakov,c Dessislava polypeptide crotamine isolated from the venom of Crotalus
Georgieva,b Banumathi durissus terrificus has been determined by single-wavelength
Sankaran,d Mario T. Murakami,e anomalous dispersion techniques and refined at 1.7 Å PDB Reference: crotamine,
Raghuvir K. Arnia and Christian resolution. The structure reveals distinct cationic and hydro- 4gv5
phobic surface regions that are located on opposite sides of
Betzelb*
the molecule. This surface-charge distribution indicates its
possible mode of interaction with negatively charged phos-
a
Multi User Center for Biomolecular Innovation, pholipids and other molecular targets to account for its diverse
Department of Physics, São Paulo State pharmacological activities. Although the sequence identity
University, UNESP/IBILCE, C. Postal 136,
between crotamine and human -defensins is low, the three-
15054-000 São José do Rio Preto-SP, Brazil,
b
Institute of Biochemistry and Molecular dimensional structures of these functionally related peptides
Biology, Hamburg University, Martin-Luther- are similar. Since crotamine is a leading member of a large
King Platz 6, 20146 Hamburg, Germany, family of myotoxic peptides, its structure will provide a basis
c
Institute of Protein Research, RAS, Pushchino, for the design of novel cell-penetrating molecules.
Moscow Region 142290, Russian Federation,
d
Physical Biosciences Division, Lawrence
Berkeley National Laboratory, 1 Cyclotron
1. Introduction
Road, Berkeley, CA 94702, USA, and
e
Biosciences National Laboratory, National Crotamine, a highly basic (pI = 10.3) 42-amino-acid poly-
Center for Energy and Materials Research, peptide (molecular mass 4.8 kDa), was first isolated in 1947
Giuseppe Maximo Scolfaro 10000,
from the venom of the Brazilian rattlesnake Crotalus durissus
13083-970 Campinas-SP, Brazil
terrificus (Gonçalves & Polson, 1947). Crotamine is of high
pharmacological importance as a potent analgesic and has
Correspondence e-mail: been shown to be over 30-fold more effective than morphine
[email protected] (Giorgi et al., 1993; Mancin et al., 1998). It also selectively
inhibits and interferes with the functioning of Kv1.3 channels,
promotes the permeability of bacterial membranes (Oguiura
et al., 2011) and is considered to be a promising cell-
penetrating agent capable of accumulating in the nucleus and
in transporting DNA into replicating cells (Kerkis et al., 2004,
2010). It has been suggested that crotamine possesses the
potential to transport drugs into mammalian cells without
requiring specific receptors. More recently, it has been
demonstrated that crotamine possesses both antitumoral and
antibacterial activities (Lee et al., 2011).
Crotamine possesses three disulfide bridges (Boni-Mitake et
al., 2001) and a number of isoforms have been characterized
(Toyama et al., 2000; Ponce-Soto et al., 2007). The overall fold
of crotamine is homologous to antimicrobial peptides (AMPs)
belonging to the -defensin, -defensin and insect defensin
families (Dimarcq et al., 1998) and possessing the same
number of disulfide bridges (Hoover et al., 2001). Despite the
differences in amino-acid composition, crotamine possesses
the same structural scaffold as mammalian -defensins and
-defensins, consisting of a three-stranded -sheet core and a
framework of loops stabilized by six disulfide-linked cysteines
(Ganz et al., 1985). Both -defensins and -defensins consist of
a triple-stranded -sheet with a distinct ‘defensin’ fold (Ganz,
# 2013 International Union of Crystallography 2003). Functionally, defensins display a wide spectrum of
Printed in Singapore – all rights reserved activities and trigger diverse effects. Some of these peptides

1958 doi:10.1107/S0907444913018003 Acta Cryst. (2013). D69, 1958–1964

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Table 1 Animals), Botucatu, Brazil was isolated by a single cation-
Crystal parameters and data-collection and refinement statistics. exchange chromatography step applying a MonoS HR 10/10
Values in parentheses are for the highest resolution shell. column (Amersham Biosciences). The molecular mass and
Pt-derivative Native sequence of the amino acids were analysed by mass spectro-
scopy and single crystals suitable for X-ray diffraction data
Data collection collection were obtained after 2 d by vapour diffusion when
Beamline X12, DORIS III P14, PETRA III
Space group I212121 I212121 crotamine at a concentration of 22 mg ml1 in deionized water
Unit-cell parameters (Å) a = 66.94, b = 74.55, a = 66.92, b = 74.33, was equilibrated against a reservoir solution consisting of
c = 80.45 c = 80.19 0.2 M sodium thiocyanate, 1.9 M ammonium sulfate pH 6.1, as
Resolution (Å) 54.6–2.5 (2.60–2.50) 54.5–1.7 (1.79–1.69)
Measured reflections 36251 (1931) 139383 (13889) described in detail previously (Coronado et al., 2012).
Completeness (%) 99.4 (75.2) 97.7 (85.3)
Averaged multiplicity 5.3 (2.5) 6.3 (5.0) 2.2. X-ray data collection
Average I/(I) 26.0 (5.7) 20.8 (1.1)
Rmerge† (%) 3.6 (16.7) 3.7 (14.2) Since attempts to solve the structure of crotamine by
Structure solution (AutoSol)
No. of sites 3
molecular replacement using either the NMR-derived coor-
Skew‡ 0.16 dinates of crotamine (PDB entries 1h5o and 1z99; Nicastro et
CORRr.m.s.§ 0.88 al., 2003; Fadel et al., 2005) or the defensin structures known to
Figure of merit (FOM) 0.36
Estimated map CC 0.56
date were unsuccessful, SAD (single-wavelength anomalous
Structure building (AutoBuild) dispersion) was applied to solve the phase problem. A native
Residues built 99 data set was collected to 1.7 Å resolution on the EMBL
Rwork (%) 33.1
Rfree (%) 37.3
beamline P14 at PETRA III (DESY/Hamburg). The native
Map CC 0.75 X-ray diffraction data were integrated, processed and scaled
Refinement using the XDS software (Kabsch, 2010). A suitable heavy-
Resolution (Å) 10.0–1.7
R factor (%) 16.6 (17.1)
metal derivative was obtained by soaking crystals for
Free R factor (%) 22.5 (22.9) approximately 12 h in a 0.1 M potassium hexachloroplatinate
Overall B factor (Å2) 33.3 (K2PtCl6) solution (Heavy Atom Screens; Hampton
R.m.s. deviations
Bond lengths (Å) 0.03
Research). Prior to data collection, crystals were flash-cooled
Bond angles ( ) 2.85 in a nitrogen-gas stream at 100 K. MAD (multi-wavelength
Ramachandran plot, residues in (%) anomalous dispersion) data were collected on the EMBL
Most favoured region 95.8
Additionally allowed region 4.2
beamline X12 at DORIS III (DESY/Hamburg) at the peak,
No. of molecules inflection and high-energy points of the Pt fluorescence
Protein 3 spectrum. However, the data from the peak wavelength
Water 65
Sulfate ions 3
displayed a very clear anomalous signal and the SAD tech-
Thiocyanate ions 4 nique was used. Anomalous data reduction and determination
Glycerol 4 of the space group and unit-cell parameters were carried out
P P P P with the iMOSFLM software (Battye et al., 2011). The data-
† Rmerge = hkl i jIi ðhklÞ hIðhklÞij= hkl i Ii ðhklÞ, where hI(hkl)i is the mean
intensity of the observations Ii(hkl) of reflection hkl. ‡ Deviation from a Gaussian collection statistics are presented in Table 1.
distribution. § Correlation of a local r.m.s. density.

2.3. Model building and refinement

possess anti-Gram-positive activities and participate in anti-
bacterial defence reactions (Cociancich et al., 1993). Phases were determined by SAD using the program Phaser
Although crotamine was isolated more than 60 years ago in the PHENIX software suite (Adams et al., 2010) at 2.5 Å
resolution by exploiting the anomalous signal of platinum ions.
(Gonçalves & Polson, 1947), it has been extremely recalcitrant
The initial electron-density map was of sufficient quality to
to crystallization, probably owing to its high intrinsic flexibility
build approximately 90% of three polypeptide chains of
as confirmed by NMR studies (Endo et al., 1989; Nicastro et al.,
crotamine present in the asymmetric unit using automated
2003; Fadel et al., 2005).
building in phenix.autobuild (Adams et al., 2010). REFMAC
In this work, we report the first crystal structure of crot-
amine, a leading member of a large family of highly basic (Murshudov et al., 2011) in combination with the inspection of
polypeptides. the electron-density maps using the program Coot (Emsley et
al., 2010) was used to complete and refine the model to 1.7 Å
resolution with an R value of 16.6% and an Rfree of 22.5%. The
final model also contains 65 solvent water molecules, four
2. Material and methods glycerol molecules, four thiocyanate ions and three sulfate
2.1. Purification of crotamine ions. Refinement statistics are presented in Table 1.
The purification and crystallization of crotamine have been
described before (Coronado et al., 2012). In summary, crot- 2.4. Small-angle X-ray scattering (SAXS)
amine from crude C. durissus terrificus venom obtained from Crotamine samples were prepared in conditions with
CEVAP (Center for the Study of Venoms and Venomous different pH values: (i) 0.05 M acetic acid pH 5.0, (ii) 0.05 M

Acta Cryst. (2013). D69, 1958–1964 Coronado et al. Crotamine 1959

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Tris–HCl pH 9.0 and (iii) pure water. SAXS data were 3. Results and discussion
collected with concentrations of 1, 2, 5 and 9.7 mg ml1. The 3.1. Overall structure
concentrations were determined at 280 nm using a NanoDrop
2000C spectrophotometer. The extinction coefficient of the Crystals of crotamine belonged to space group I212121, with
protein was calculated using the online program ProtParam unit-cell parameters a = 66.92, b = 74.33, c = 80.19 Å, and
(Gasteiger et al., 2005). SAXS data from crotamine solutions contained three molecules in the asymmetric unit. The overall
were collected on EMBL beamline P12 at PETRA III (DESY/ fold of crotamine is illustrated in Fig. 1(a). Residues Lys2–
Hamburg) at 295 K using a two-dimensional photon-counting Lys7 form a single -helical turn which flanks a two-stranded
PILATUS 2M pixel X-ray detector (DECTRIS). All data sets antiparallel -sheet formed by residues Gly9–Pro13 (1) and
were normalized to the incident-beam intensity and corrected Trp34–Lys38 (2) located in the core of the molecule. A short
for detector response, and scattering of the buffer was -helical turn is formed by residues Pro20–Ser23. The poly-
subtracted using ATSAS (Petoukhov et al., 2007). peptide is stabilized by three disulfide bonds: Cys4–Cys36,
Cys11–Cys30 and Cys18–Cys37. The disulfide bridge Cys4–
Cys36 anchors the first -helical segment to 2. 1 and 2 are
Table 2
connected by a flexible loop Lys14–Leu19, and helical turn 2
Hydrogen-bond contacts of crotamine molecules in the asymmetric unit. is connected to 2 by a more extended and flexible loop
formed by residues Asp24–Arg33. Overall, the topology can
A B C Distance (Å)
be classified as 1122. The -sheet is stabilized by
Glu15 OE1 Lys35 NZ 3.27 hydrogen bonds between strands 1 and 2, involving residues
Glu15 OE1 Cys18 N 2.91
Asp24 O Lys14 NZ 3.11
His10–Cys37 and Phe12–Lys35, and hydrogen bonds between
Lys35 NZ Glu15 OE1 2.95 strand 2 and 2, formed by Ser23–Lys38. Two hydrogen
Cys18 N Glu15 OE1 2.89 bonds connect 2 to the C-terminal -turn.
Lys14 NZ Pro21 O 3.00
Lys14 NZ Asp24 O 2.79
As crotamine is relatively small, all charged as well as
Cys11 O Arg31 NH1 2.82 hydrophobic residues are exposed to the solvent, a circum-
Cys11 O Arg31 NH2 2.97 stance that makes crotamine unusually ‘sticky’. It is likely that
Arg31 NH2 Tyr1 OH 2.64
these electrostatic and hydrophobic forces on the surface, in
combination with a disulfide-stabilized
molecular scaffold, enable crotamine or
crotamine oligomers to complex with
target proteins. Most hydrophobic resi-
dues are located on one side of the
molecule and the positively charged
residues Lys2, Lys6, Lys7, Lys14, Lys16,
Lys27, Lys35, Lys38, Lys39, Arg31 and
Arg33 are clustered on the opposite
side, as shown in Fig. 1(b). These resi-
dues are exposed on the surface of
crotamine and form distinct hydro-
phobic and cationic regions which are
positioned roughly on opposite sides of
the amphiphilic molecule, as shown in
Fig. 1(c).
Superposition with the crotamine
NMR structure (Nicastro et al., 2003;
Fadel et al., 2005) resulted in a mean C
r.m.s.d. value of 2.12 Å, indicating a
relatively high spatial deviation
between the NMR and crystal struc-
tures, and higher flexibility of the NMR
solution structure.

3.2. The quaternary arrangement

Both SAXS and dynamic light-
Figure 1 scattering measurements indicated that
(a) Structure of crotamine. The N- and C-termini and the cysteine residues are labelled. (b) The
the protein was monomeric in solution
highly hydrophobic residues in pink are located on one side of the molecule and the positively
charged residues in blue are on the opposite side. (c) Space-filling presentation of crotamine, (Coronado et al., 2012). In the crystal
highlighting the well defined amphipathic surface region in blue and pink. structure three molecules of crotamine

1960 Coronado et al. Crotamine Acta Cryst. (2013). D69, 1958–1964

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are present in the asymmetric
unit (Fig. 2), which is stabilized by
a number of intermolecular
contacts involving main-chain
and side-chain atoms. In the
crystal structure chains A–B, A–C
and B–C bury 353, 261 and
121 Å2, respectively. This corre-
sponds to approximately 6–10%
of the overall surface area of each
monomer. A careful inspection of
interactions stabilizing the trimer
in the crystal showed the
presence of two sulfate ions in the
interface of chains A and C, close
to residues Cys11 and His10 of
chain C and Lys16, Phe12 and
Pro13 of chain A. In addition,
four glycerol and three thiocya-
nate molecules were identified at
almost equivalent positions in the
interface regions of chains A–B,
B–C and C–A, forming hydrogen-
bonding, van der Waals and
hydrophobic interactions stabi-
lizing the trimer. All inter-
molecular interactions are
summarized in Table 2.

3.3. Structural similarities

Figure 2 between crotamine and
(a) Cartoon plot of the crotamine trimer. (b) Surface-charge distribution in two orientations. antimicrobial peptides
The high positively charged
surface permits us to hypothesize
that crotamine can interact elec-
trostatically with the negatively
charged surface of membranes
with the potential to induce the
formation of gaps, through which
ions and/or other molecules can
diffuse.
In Fig. 3(a) and Table 3 a
sequence comparison and struc-
tural characteristics of homo-
logous antimicrobial (AMP) and
antimicrobial-like (defensin-like)
peptides of different origin are
shown. The defensin-like poly-
peptides share relatively low
sequence identities in the range
Figure 3
(a) Sequence alignment of antimicrobial peptides. Conserved Cys residues are indicated in dark grey. 15–35%; however, they have a
Crotamine (PDB entry 4gv5), defensin-like peptide 2 (PDB entry 1d6b), heliomicin (PDB entry 1i2u), toxin homologous secondary-structural
III (PDB entry 1lqq), Eucommia antifungal peptide 2 (PDB entry 1p9z), charybdotoxin (PDB entry 2crd), arrangement of -helices, -
human -defensin 1 (PDB entry 1iju), human -defensin 2 (PDB entry 1fd4), human -defensin 3 (PDB
sheets and random coils, as well
entry 1kj6) and human -defensin 1 (PDB entry 2pm1). (b) Superposition of crotamine (cartoon plot in
light grey) with hD-1 (PDB entry 1iju, pink), hD-2 (PDB entry 1fd4, red) and hD-3 (PDB entry 1kj6, as the conservation of six cysteine
cyan). The corresponding C r.m.s.d. values are 1.8, 1.8 and 2.6 Å, respectively. residues forming three disulfide

Acta Cryst. (2013). D69, 1958–1964 Coronado et al. Crotamine 1961

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Table 3
Comparison of the amino-acid sequence of crotamine with -defensin, -defensin and defensin-like peptides.
Amino-acid sequences are given in one-letter code.
Disulfide PDB
AMP Length Amino-acid sequence Secondary structure bridges Organism code

CRO 42 YKQCHKKGGHCFPKEKICLPPSSDFGKMDCRWR- -Helix aligned to antiparallel 3 Crotalus durissus 4gv5

WKCCKKGSG two-stranded -sheet terrificus
EAFP-2 30–43 ETCASRCPRPCNAGLCCSIYGYCGSGAAYCGAGN- Two -helices aligned to three- 5 Eucommia ulmoide 1p9z
CRCQCRG stranded -sheet random coils
CHTX 30–40 EFTNVSCTTSKECWSVCQRLHNTSRGKCMNKKC- Small three-strand -sheet, 3 Leiurus quinquestriatus 2crd
RCYS -helix, random coil hebraeus
HEL 44 DKLIGSCVWGAVNYTSDCNGECKRRGYKGGHC- -Helix, three-stranded antiparallel 3 Heliothis virescen 1i2u
GSFANVNCWCET -sheet, random coil
Toxin III 64 VRDAYIAKNYNCVYECFRDSYCNDLCTKNGASS- -Helix, three-stranded antiparallel 4 Leiurus quinquestriatus 1lqq
GYCQWAGKYGNACWCYALPDNVPIRVPGKCH -sheet, random coil hebraeus
DLP-2 42 IMFFEMQACWSHSGVCRDKSERNCKPMAWTYCE- Small -helix, two-stranded -sheet, 3 Ornithorhynchus 1d6b
NRNQKCCEY random coils anatinus
HD-1 36 DHYNCVSSGGQCLYSACPIFTKIQGTCYRGKAK- -Helix, three-stranded -sheet, 3 Homo sapiens 1iju
CCK random coils
HD-2 41 GIGDPVTCLKSGAICHPVFCPRRYKQIGTCGLPGT- -Helix, three-stranded -sheet, 3 Homo sapiens 1fd4
KCCKKP random coils
HD-3 45 GIINTLQKYYCRVRGGRCAVLSCLPKEEQIGKCS- -Helix, three-stranded -sheet, 3 Homo sapiens 1kj6
TRGRKCCRRKK random coils
HNP-1 30 ACYCRIPACIAGEAAYGTCIYQGALWAFCC Three-stranded -sheet, 3 Homo sapiens 2pm1
random coils

Table 4 -defensins and crotamine, as shown in

Table 4. These distributions of charged
Summary of charged and hydrophobic residues of crotamine in comparison to other antimicrobial
peptides. residues are also typical for -defensins
SA: total surface accessibility. from other animals. In contrast, lysine
CRO HD-1 HD-2 HD-3 CHTX DLP-2 HEL Toxin III HNP-1 EAFP-2 residues are relatively rare in -defen-
sins. The predominance of positively
Arg+ 2 1 2 7 3 3 2 3 1 4 charged residues in crotamine is likely

Asp 2 1 1 — — 1 2 4 — —
Glu 1 — — 2 2 4 2 1 1 1 to facilitate electrostatic interactions
Lys+ 9 4 5 6 4 3 3 4 — — with the anionic membrane surface. The
Total 14 6 8 15 9 11 9 12 2 5 total surface accessibility of crotamine
Positive 11 5 7 13 7 6 5 7 1 4
Negative 3 1 1 2 2 5 4 5 1 1 and hDs differs, the accessible surface
Trp 2 — — — 1 2 2 2 1 — area changes occur when residues on
Tyr 1 3 1 — 1 2 2 7 3 3 the molecule surface are replaced by
Phe 2 1 1 — 1 2 1 1 1 —
SA (Å2) 3223 2808 2858 4025 3019 3888 3060 4069 2246 2997 others having large or smaller side
chains (Table 4). The -helix, the anti-
parallel -sheet and the -turn present
in the human -defensins 1–3 (Hoover
bonds (Schibli et al., 2002). An overall structural comparison et al., 2000, 2001; Schibli et al., 2002) are also conserved in
of the three known human defensin structures hD-1, hD-2 crotamine.
and hD-3 with crotamine is shown in Fig. 3(b). The corre- Fig. 4 is an overview highlighting the structural similarities
sponding C r.m.s.d. values are 1.8, 1.8 and 2.6 Å, respectively. and differences of defensin-like polypeptides. In all structures
It is obvious that, despite the moderate sequence identity, the basic secondary-structural elements and disulfide bridges,
evolutionary selective pressures have favoured a similar a feature initially attributed to polypeptides classified as
overall three-dimensional structure and fold of these func- defensins, are conserved. However, toxin III and Eucommia
tionally related peptides. The predominance of functionally antifungal peptide 2 (EAFP-2) have four and five disulfide
relevant positively charged residues (Table 4) is a common bonds, respectively. Among the antimicrobial polypeptides
feature of crotamine and defensins. Most probably this facil- with six cysteines the -defensin and -defensin group is the
itates the electrostatic interactions with the anionic membrane best characterized to date. They are widely distributed in
surface. different phyla, including plants, insects, arthropods and
Despite high overall structural conservation, some vertebrates (Ganz, 2004; Lehrer et al., 1993). The polypeptide
physicochemical differences between human -defensins and defensin-like peptide 2 (DLP-2) isolated from platypus
crotamine have been addressed as likely determinants of the (Ornithorhynchus anatinus) venom (Torres et al., 2000) shares
observed functional differences (Yount et al., 2009). The 34% sequence identity with crotamine but displays no anti-
arginine and lysine content varies between the three human microbial activity. Heliomicin (HEL), which is known to be an

1962 Coronado et al. Crotamine Acta Cryst. (2013). D69, 1958–1964

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Figure 4
Structure comparison of crotamine with homologous antimicrobial and antimicrobial-like peptides from different organisms in two orientations. Column
1, the -core domain is shown in orange. Column 2, hydropathy plot overlay: dark orange/hydrophobic; light orange/intermediate; blue/hydrophilic.
Column 3, surface charge: blue, basic (Arg, Lys); red, acidic (Asp, Glu). Row A, crotamine from C. durissus terrificus venom (PDB entry 4gv5); row B,
defensin-like peptide 2 (1d6b); row C, heliomicin (1i2u); row D, toxin III (1lqq); row E, Eucommia antifungal peptide 2 (1p9z); Row F, charybdotoxin
(2crd); row G, human -defensin 1 (1iju); row H, human -defensin 2 (1fd4); row I, human -defensin 3 (1kj6); row J, human -defensin 1 (2pm1).

Acta Cryst. (2013). D69, 1958–1964 Coronado et al. Crotamine 1963

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This project was supported by grants from the Deutsche 1407–1409.
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(2010). Expert Opin. Investig. Drugs, 19, 1515–1525.
FAPESP, CNPq, CAPES and DAAD (PROBAL 50754442). Lamberty, M., Caille, A., Landon, C., Tassin-Moindrot, S., Hetru, C.,
MAC thanks CAPES (grant NR-8248/12-5) for the exchange Bulet, P. & Vovelle, F. (2001). Biochemistry, 40, 11995–12003.
fellowship. DG thanks the Alexander von Humboldt Foun- Landon, C., Cornet, B., Bonmatin, J. M., Kopeyan, C., Rochat, H.,
dation (AvH), Bonn, Germany for providing a Research Vovelle, F. & Ptak, M. (1996). Eur. J. Biochem. 236, 395–404.
Fellowship. The Berkeley Center for Structural Biology is Lee, J.-Y., Choi, Y.-S., Suh, J.-S., Kwon, Y.-M., Yang, V. C., Lee, S.-J.,
Chung, C.-P. & Park, Y.-J. (2011). Int. J. Cancer, 128, 2470–2480.
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National Institute of General Medical Sciences and the Immunol. 11, 105–128.
Howard Hughes Medical Institute. The Advanced Light Mancin, A. C., Soares, A. M., Andrião-Escarso, S. H., Faça, V. M.,
Source is supported by the US Department of Energy under Greene, L. J., Zuccolotto, S., Pelá, I. R. & Giglio, J. R. (1998).
Contract No. DE-AC02-05CH11231. We would like to thank Toxicon, 36, 1927–1937.
Murshudov, G. N., Skubák, P., Lebedev, A. A., Pannu, N. S., Steiner,
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