Báo cáo khoa học: Cupiennin 1a, an antimicrobial peptide from the venom of the neotropical wandering spider Cupiennius salei, also inhibits the formation of nitric oxide by neuronal nitric oxide synthase pptx

Cupiennin 1a, an antimicrobial peptide from the venom
of the neotropical wandering spider Cupiennius salei,
also inhibits the formation of nitric oxide by neuronal
nitric oxide synthase
Tara L. Pukala
1
, Jason R. Doyle
2
, Lyndon E. Llewellyn
2
, Lucia Kuhn-Nentwig
3
, Margit A. Apponyi
1
,
Frances Separovic
4
and John H. Bowie
1
1 Department of Chemistry, The University of Adelaide, Australia
2 Institute of Marine Science, Townsville, Queensland, Australia
3 Zoological Institute, University of Bern, Switzerland
4 School of Chemistry, Bio21 Institute, University of Melbourne, Australia
The neotropical wandering spider Cupiennius salei is a
large, nocturnal hunting spider distributed throughout
Central America, located as far north as Veracruz state
in Mexico and extending to Honduras in the south. It
is restricted to altitudes ranging from 200 to 1250 m,
and resides in the tropical rain forests of this region
[1]. The spider is brown, with small, light spots on the
abdomen and many dark longitudinal stripes, predom-

inantly on the carapace. The legs, patella and femurs
are also striped with lighter circles, and the underbody
is red–yellow with thin black vertical stripes under the
abdomen. Females can reach up to 35 mm in body
length and have a 100 mm leg span, whereas males are
typically smaller and less brightly coloured [1].
The venom of C. salei is a natural insecticide, caus-
ing a rapid and dose-dependent paralysis of prey up to
a critical lethal dose [1]. Three classes of molecules
comprise the venom, and can be categorized on the
basis of molecular weights. The first group consists of
low molecular weight compounds, including ions, free
amino acids, amines and polyamines [2]. The second
group includes mainly proteins with masses between 25
and 27 kDa. Among these, a highly active hyaluroni-
dase has been reported, which is a spreading factor
used to accelerate toxin transport into the tissue [2].
Even under extreme test conditions, only very low lev-
els of proteolytic enzymes are observable. The final
group comprises peptides with masses generally in the
Keywords
cupiennin 1a; Cupiennius salei; neuronal
nitric oxide synthase activity; two-
dimensional NMR
Correspondence
J. H. Bowie, Department of Chemistry, The
University of Adelaide, Adelaide, SA 5005,
Australia
Fax: +61 08 830 34358
Tel: +61 08 830 3567

E-mail:
(Received 22 August 2006, revised 17 Janu-
ary 2007, accepted 1 February 2007)
doi:10.1111/j.1742-4658.2007.05726.x
Cupiennin 1a (GFGALFKFLAKKVAKTVAKQAAKQGAKYVVNKQ-
ME-NH
2
) is a potent venom component of the spider Cupiennius salei.
Cupiennin 1a shows multifaceted activity. In addition to known antimicro-
bial and cytolytic properties, cupiennin 1a inhibits the formation of nitric
oxide by neuronal nitric oxide synthase at an IC
50
concentration of
1.3 ± 0.3 lm. This is the first report of neuronal nitric oxide synthase inhi-
bition by a component of a spider venom. The mechanism by which cupi-
ennin 1a inhibits neuronal nitric oxide synthase involves complexation with
the regulatory protein calcium calmodulin. This is demonstrated by chem-
ical shift changes that occur in the heteronuclear single quantum coherence
spectrum of
15
N-labelled calcium calmodulin upon addition of cupien-
nin 1a. The NMR data indicate strong binding within a complex of 1 : 1
stoichiometry.
Abbreviations
Ca
2+
-CaM, calcium calmodulin; HSQC, heteronuclear single quantum coherence; NOS, nitric oxide synthase; nNOS, neuronal nitric oxide
synthase.
1778 FEBS Journal 274 (2007) 1778–1784 ª 2007 The Authors Journal compilation ª 2007 FEBS
range 2–8 kDa. These include: (a) the neurotoxins

CSTX-1, CSTX-9 and CSTX-13 [3,4]; and (b) the anti-
microbial and cytolytic cupiennins 1a to 1d [5–8]. The
sequence of cupiennin 1a is GFGALFKFLAKK-
VAKTVAKQAAKQGAKYVVNKQME-NH
2
.
The cupiennins are membrane-active wide-spectrum
antimicrobials; the most stable structure of cupien-
nin 1a (as determined by two-dimensional NMR meth-
ods in trifluoroethanol ⁄ water, 1 : 1 [9]) is the hinged
structure shown in Fig. 1 (the hinge occurs at Gly25).
It has been suggested that the role of these antimicro-
bial peptides in the venom of C. salei may be two-fold:
(a) the cheliceral claws, which first penetrate the prey,
are heavily exposed to external pathogens, and thus
the antibacterial peptides may be involved in protec-
tion of the venom apparatus against infection; and
(b) the cytolytic activity of these peptides may afford
the neurotoxins better access to their intercellular tar-
gets [7].
After the secondary structure determination of the
strongly basic and hinged peptide cupiennin 1a was
finalized (Fig. 1), it was clear that this peptide showed
some structural features in common with certain
amphibian peptides that inhibit the formation of NO
by neuronal nitric oxide synthase (nNOS) [10]. These
particular amphibian peptides (e.g. the caerins 1 and
splendipherin [11]), are basic and hinged, and inhibit
the operation of nNOS by complexing with the regula-
tory protein calcium calmodulin (Ca

2+
-CaM).
In this article, we report that cupiennin 1a also com-
plexes with Ca
2+
-CaM, and is one of the more active
of the known peptide inhibitors of nNOS.
Results
Cupiennin 1a was tested for the ability to inhibit
nNOS, using an assay that measures the conversion of
[
3
H]arginine to [
3
H]citrulline by this enzyme [10]. Cupi-
ennin 1a produces a dose-dependent inhibition of
nNOS: the IC
50
value and Hill slope [12] are
1.3 ± 0.3 lm (5.1 ± 1.1 lgÆmL
)1
) and 3.5 ± 1.0,
respectively. These values are comparable to those of
amphibian peptides, which inhibit nNOS by complex-
ing with the regulatory protein Ca
2+
-CaM [10,11].
A
15
N heteronuclear single quantum coherence

(HSQC) titration was performed to determine whether
cupiennin 1a interacts with CaM to inhibit the action
of nNOS. Increasing quantities of unlabelled cupien-
nin 1a were added to
15
N-labelled Ca
2+
-CaM, and a
high-resolution
15
N HSQC spectrum was recorded
after each addition. The chemical shift changes were
then tracked by overlaying each of the spectra, as can
be seen in Figs 2 and 3. Chemical shift changes were
considered to be significant when they were greater
than 0.5 p.p.m. in the nitrogen dimension and greater
than 0.05 p.p.m. in the hydrogen dimension [13].
Evidence of complex formation is apparent, with the
titration series showing distinct chemical shift changes
for a large number of residues throughout the Ca
2+
-
CaM sequence. The chemical shifts do not change as a
function of concentration; rather, a second set of peaks
appear with distinct chemical shifts after addition of
only 0.4 equivalents of cupiennin 1a. Peak intensities
for the bound and unbound conformers at 0.6 equiva-
lents of peptide are comparable, suggesting a 1 : 1 stoi-
Fig. 1. The lowest calculated potential
energy structure of cupiennin 1a in

d
3
-trifluoroethanol ⁄ water (1 : 1).
T. Pukala et al. Cupiennin 1a from Cupiennius salei
FEBS Journal 274 (2007) 1778–1784 ª 2007 The Authors Journal compilation ª 2007 FEBS 1779
chiometry and providing evidence of slow exchange
binding. The peptide was fully bound and gave rise to
a completely new set of protein chemical shifts at a
1 : 1 molar concentration, with continued addition of
peptide to 2 : 1 equivalents having no further effect on
the chemical shifts (data not shown). The peak intensi-
ties for the bound and unbound conformations are
approximately equal upon full saturation, further indi-
cating that the complex is stable on the NMR time-
scale and exists in a slow exchange regime.
Selected resonances in the HSQC spectrum were
assigned on the basis of the chemical shifts reported
previously for unbound Ca
2+
-CaM [14,15]. No
attempt was made to assign either unbound resonances
in the NMR spectra of Ca
2+
-CaM, or of the fully
bound complex, in regions where the density of signals
would result in ambiguity. Even so, distinct chemical
shift changes occur for a large number of Ca
2+
-CaM
resonances, and this can be seen from the selected

labelled peaks shown in Fig. 3 (e.g. T5, G25, I27, T29,
G61, G98, L116, T117, N137 and A147). This is con-
sistent with a substantial change in Ca
2+
-CaM confor-
mation following complexation with cupiennin 1a.
Discussion
NO is unique among biological signals for its rapid
diffusion, ability to permeate cell membranes, and
intrinsic instability, properties that eliminate the need
for extracellular NO receptors or targeted NO degra-
dation [16,17]. NO is produced by three NOSs (in ver-
tebrates), which oxidize l-arginine to NO and
citrulline, thereby controlling NO distribution and
Fig. 2. 15N HSQC spectra of CaM in the absence of cupiennin 1a
(red), and with the addition of cupiennin 1a in a 1 : 1 molar ratio
(purple).
Fig. 3. Partial overlaid
15
N HSQC spectra for the titration of Ca
2+
-
CaM with cupiennin 1a. The peptide ⁄ protein ratio is indicated.
Cupiennin 1a from Cupiennius salei T. Pukala et al.
1780 FEBS Journal 274 (2007) 1778–1784 ª 2007 The Authors Journal compilation ª 2007 FEBS
concentration. The isoforms of NOS are homodimers
with subunits of 130–160 kDa, differing in amino acid
sequence identity, but sharing an overall three-compo-
nent construction, namely: (a) an N-terminal catalytic
oxygenase domain that binds heme, tetrahydrobiopter-

in and l-arginine; (b) a C-terminal reductase domain
that binds FMN, FAD and NADPH; and (c) an inter-
vening CaM-binding region that regulates electronic
communication between the oxygenase and reductase
domains [16,17]. NOS enzymes are found in most life-
forms [16,17], including bacteria [18–21] and insects
[22–24].
Ca
2+
-CaM is a dumbbell-shaped 148-residue protein
containing two terminal units, each of which may con-
tain two Ca
2+
.Ca
2+
-CaM is required for the activa-
tion of nNOS: it is a regulatory protein that acts as an
electron shuttle and Ca
2+
transporter. It also alters
the conformation of the reductase domain, allowing
reactions to proceed at the heme site [17]. nNOS-active
peptides interfere with communication between
Ca
2+
-CaM and nNOS, because the complex formed
between the active peptide and Ca
2+
-CaM has a dif-
ferent shape from that of Ca

2+
-CaM [10,11,25,26],
and therefore adversely affects binding of CaM to the
Ca
2+
-CaM-binding domain of nNOS. CaM is not
only essential for the operation of nNOS and the other
NOS isoforms, but is also the regulatory protein for a
variety of other enzymes, including kinases [27–29].
Two peptide–CaM binding modes have been identi-
fied by NMR studies [30]. These are shown in Fig. 4. In
the first, Ca
2+
-CaM adopts a compact, globular shape,
with the peptide engulfed in a hydrophobic channel
formed by the two terminal domains [25,30–32]. The
example shown in Fig. 4A is that of the 26-residue pep-
tide fragment of skeletal myosin light chain kinase, with
residues 3–21 encompassed within Ca
2+
-CaM [25]: this
type of structure is also adopted by Ca
2+
-CaM when it
binds to the CaM-binding domain of endothelial nitric
oxide synthase (eNOS) [33]. The second example is
when the C-terminal lobe of Ca
2+
-CaM binds part of
the target peptide. This is shown in Fig. 4B for the 20-

residue binding domain of the plasma membrane Ca
2+
pump ⁄ Ca
2+
-CaM complex, where the first 12 residues
of the peptide are encompassed by the C-terminal end
of Ca
2+
-CaM [26].
Previous studies have indicated that binding of pep-
tides to Ca
2+
-CaM requires the peptide: (a) to adopt
an amphipathic a-helical conformation when binding
to CaM [10,11,25,26]; (b) be positively charged
[10,11,25,26]; and (c) display large hydrophobic resi-
dues in conserved positions, which point to one face in
a presumed helical conformation [30]. It has been pro-
posed that the extent of hydrophobic anchoring deter-
A
B
Fig. 4. (A) Myosin light-chain kinase ⁄ Ca
2+
-CaM complex [25]. (B)
Binding domain of the plasma membrane Ca
2+
pump ⁄ Ca
2+
-CaM
complex [26].

T. Pukala et al. Cupiennin 1a from Cupiennius salei
FEBS Journal 274 (2007) 1778–1784 ª 2007 The Authors Journal compilation ª 2007 FEBS 1781
mines which of the two binding modes (Fig. 4A,B) is
adopted by the complex [30]: this is supported by
small-angle X-ray scattering experiments [34].
Cupiennin 1a conforms to all of these prerequisites.
It is unstructured in water [6], but adopts a helical struc-
ture in the membrane mimicking the solvent d
3
-trifluor-
oethanol ⁄ water (1 : 1) [9], as shown in Fig. 1. The large
number of lysine residues gives the peptide an overall
charge of + 8. As the N-terminal helix of cupiennin 1a
is more amphipathic than the C-terminus, and also has
a greater positive charge, it is reasonable to suppose
that Ca
2+
-CaM is more likely to bind to this region of
the peptide. Furthermore, the hydrophobic face of the
amphipathic N-terminal helix of cupiennin 1a has a sig-
nificant number of long-chain aliphatic or aromatic resi-
dues (L, V and F) available to act as hydrophobic
anchors. Given the length of the first helix of cupien-
nin 1a, and the number of hydrophobic anchors avail-
able, it seems likely that the cupiennin 1a ⁄ Ca
2+
-CaM
complex is analogous to the structure shown in Fig. 4A.
In such a case, some 20 residues of cupiennin 1a could
be situated within the globular Ca

2+
-CaM.
This proposal is consistent with the data shown in
Figs 2 and 3, which indicate that chemical shift chan-
ges occur throughout the CaM sequence, including the
C-terminal and N-terminal domains. This means that a
substantial change in conformation occurs for the
regulatory protein upon binding of cupiennin 1a to
Ca
2+
-CaM, indicating that the complex forms with a
significantly different structure, rather than localized
structural differences at the binding interfaces. Analy-
sis of the chemical shift changes for those resonances
that are readily assigned (Fig. 3) is also consistent with
those reported for the structure shown in Fig. 4A [35].
Conclusion
Cupiennin 1a is a potent venom component of C. salei
with multifaceted activity, including antimicrobial
activity and inhibition of nNOS. We propose that the
inhibition of nNOS involves the formation of a glob-
ular Ca
2+
-CaM ⁄ cupiennin 1a complex, which prevents
Ca
2+
-CaM from occupying the CaM-binding domain
of nNOS. This will drastically influence numerous pro-
cesses that rely on NO as a neurotransmitter in both
prokaryotic and eukaryotic cells. CaM is not only

essential for the operation of nNOS and the other
NOS isoforms, but is also the regulatory protein for a
variety of kinase phosphorylating enzymes and adeny-
late cyclase [28], and is involved in regulation of the
eukaryote cytoskeleton [28]. The likelihood is that
cupiennin 1a will interfere with many cellular functions
simultaneously, causing maximum inconvenience and
deterrence to any attacker or pathogen, and also assist
with the rapid immobilization of prey.
Experimental procedures
nNOS bioactivity testing
nNOS inhibition testing was conducted by the Australian
Institute of Marine Science (Townsville, Australia). Inhibi-
tion was measured and analyzed by monitoring the conver-
sion of [
3
H]arginine to [
3
H]citrulline by nNOS, using a
method reported previously [10].
15
N HSQC titration
15
N-labelled Ca
2+
-CaM was prepared by a method based
on that of Elshorst et al. [26]. Briefly, CaM was expressed
in Escherichia coli strain BL21(DE3), using the expression
vector pET28 (Novagen, Madison, WI, USA). CaM expres-
sion was induced by addition of isopropyl thio-b-d-galacto-

side (0.1 mm), cells were harvested and lysed by sonication,
and CaM was purified from the supernatant using anion
exchange and size exclusion chromatography.
Samples used for the titration series contained
15
N-labelled
Ca
2+
-CaM (3.16 mg, 1.89 · 10
)7
mol), potassium chloride
(100 mm), calcium chloride (6.2 mm) and 10% D
2
O in aque-
ous solution at pH 6.3. Sodium azide (0.02%) was added as
a preservative [14]. Cupiennin 1a (1.44 mg, 3.79 · 10
)7
mol)
was dissolved in water, adjusted to pH 6.3 using sodium
hydroxide, and then divided into aliquots such that succes-
sive additions would achieve total peptide concentrations of
0.2, 0.4, 0.6, 0.8, 1 and 2 molar equivalents. The aliquots were
then lyophilized, and the dried peptide portions added to the
CaM sample in sequence. The pH was readjusted back to 6.3
with the addition of small quantities of hydrochloric acid or
sodium hydroxide solutions as required.
Spectra were recorded using a Varian (Palo Alto, CA,
USA) Inova-600 NMR spectrometer, with a
1
H frequency

of 600 MHz and a
13
C frequency of 150 MHz. Experiments
were conducted at 25 °C, and referenced to sodium 3-(tri-
methylsilyl)propane-1-sulfonate at 0 p.p.m. in
1
H, whereas
the
15
N dimension was centred at 120 p.p.m. relative to
NH
3
as 0 p.p.m. The standard gNhsqc pulse sequence from
the VNMR library was used, with 256 increments, each
comprising 16 transients, acquired over 2048 data points. A
spectral width of 7197.5 Hz was used in the
1
H dimension,
and a spectral width of 2200 Hz in the
15
N dimension. The
resultant spectra were processed using nmrpipe [36], and
viewed with sparky software (version 3.111) [37].
Acknowledgements
J. H. Bowie and F. Separovic thank the Australian
Research Council for the financial support of this
Cupiennin 1a from Cupiennius salei T. Pukala et al.
1782 FEBS Journal 274 (2007) 1778–1784 ª 2007 The Authors Journal compilation ª 2007 FEBS
project. T. L. Pukala and M. A. Apponyi acknowledge
the award of postgraduate scholarships. The pET28

vector containing the calmodulin gene was a generous
gift from Dr Joachim Krebs of the Max Plank Insti-
tute for Biophysical Chemistry, Go
¨
ttingen, Germany.
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