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: john.bowie@adelaide.edu.au (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. References 1 Barth FG (2002) A Spider’s World. Senses and Beha- viour. Springer-Verlag, Berlin. 2 Kuhn-Nentwig L, Schaller J & Nentwig W (2004) Biochemistry, toxicology and ecology of the venom of the spider Cupiennius salei (Ctenidae). Toxicon 43, 543–553. 3 Kuhn-Nentwig L, Schaller J & Nentwig W (1994) Puri- fication of toxic peptides and the amino acid sequence of CSTX-1 from the multicomponent venom of Cupien- nius salei (Ctenidae). Toxicon 32, 287–302. 4 Wullschleger B, Kuhn-Nentwig L, Tromp J, Kanpfer U, Schaller J, Schurch S & Nentwig W (2004) CSTG-13, a highly synergistically acting two-chain neurotoxic enhancer in the venom of the spider Cupiennius salei (Ctenidae). Proc Natl Acad Sci USA 101, 11251–11256. 5 Haeberli S, Kuhn-Nentwig L, Schaller J & Nentwig W (2000) Characterisation of antibacterial activity of pep- tides isolated from the venom of the spider Cupiennius salei (Ctenidae). Toxicon 38, 373–380. 6 Kuhn-Nentwig L, Mu ¨ ller J, Schaller J, Walz A, Dathe M & Nentwig W (2002) Cupiennin 1, a new family of highly basic antimicrobial peptides in the venom of Cupiennius salei (Ctenidae). J Biol Chem 277, 11208–11216. 7 Kuhn-Nentwig (2003) Antimicrobial and cytolytic peptides of venomous arthropods. Cell Mol Life Sci 60, 2651–2668. 8 Wullschleger B, Nentwig W & Kuhn-Nentwig L (2005) Spider venom: enhancement of venom efficacy mediated by different synergistic strategies in Cupiennius salei. J Expt Biol 208, 2115–2121. 9 Pukala TL, Boland M, Gehman JD, Kuhn-Nentwig L, Separovic F & Bowie JH (2006) Solution structure and interaction of cupiennin 1a, a spider venom peptide, with phospholipid bilayers. Biochemistry in press. 10 Doyle JR, Llewellyn LE, Brinkworth CS, Bowie JH, Wegener KL, Rozek T, Wabnitz PA, Wallace JC & Tyler MJ (2002) Amphibian peptides that inhibit nNOS. The isolation of lesueurin from the skin secretion of the Australian Stony Creek Frog Litoria lesueuri. Eur J Bio- chem 269, 100–109. 11 Pukala TL, Bowie JH, Maselli VM, Musgrave IF & Tyler MJ (2006) Host-defence peptides from the glandu- lar secretions of amphibians: structure and activity. Nat Prod Rep 23, 368–393. 12 Fersht A (1985) Cooperative ligand binding and allos- teric interactions in enzyme structure and mechanism. In Enzyme Structure and Mechanism, pp. 208–225. Free- man WH, New York, NY. 13 Schon O, Friedler A, Freund S & Fersht AR (2004) Binding of p53-derived ligands to MDM2 induces a variety of long range conformational changes. J Mol Biol 336, 197–202. 14 Ikura M, Kay LE & Bax A (1990) A novel approach for sequential assignment of 1 H, 13 C and 15 N spectra of larger proteins: heteronuclear triple-resonance three- dimensional NMR spectroscopy. Application to calmo- dulin. Biochemistry 29, 4659–4667. 15 Torizawa T, Shimizu M, Taoka M, Miyano H & Kainosho M (2004) Efficient production of isotopically labelled proteins by cell free synthesis: a practical proto- col. J Biomol NMR 30, 311–325. 16 Kerwin JF, Lancaster JR & Feldman PL (1995) Nitric oxide: a new paradigm for secondary messengers. J Med Chem 38, 4344–4362. 17 Stuehr DJ & Ghosh S (2000) Nitric Oxide. Handbook of Experimental Pharmacology (Mayer B, ed.), pp. 33–70. Springer-Verlag, Berlin. 18 Choi WS, Chang MS, Han JW, Hong SY & Lee HW (1997) Identification of NOS in Staphylococcus aureus. Biochem Biophys Res Commun 237, 554–558. 19 Morita H, Yoshikawa H, Sakata R, Nagata Y & Tanaka H (1997) Synthesis of nitric oxide from the two equivalent quanidino nitrogens of L-arginine by Lacto- bacillus fermentum. J Bacteriol 179, 7812–7815. 20 Choi WS, Seo DW, Chang MS, Han JW, Paik WK & Lee HW (1998) Methyl esters of L-arginine and N-nitro-L-arginine induce NOS in Staphylococcus aureus. Biochem Biophys Res Commun 246, 431–435. 21 Er H, Turkoz Y, Ozerol IH & Uzmez E (1998) Effect of NOS inhibition in experimental Pseudomonas keratitis in rabbits. Eur J Opthalmol 8, 137–141. 22 Muller U (1994) Ca 2+ calmodulin-dependent nitric oxide synthase in Apis mellifera and Drosophila melano- gaster. Eur J Neurosci 6, 1362–1370. 23 Regulski M & Tully T (1995) Molecular and biochem- ical characterization of dNOS: a Drosophila Ca 2+ cal- modulin-dependent nitric oxide synthase. Proc Natl Acad Sci USA 92, 9072–9076. 24 D’yakonova VE & Krushinskii AL (2006) Effects of an NOS inhibitor on aggressive and sexual behaviour in male crickets. Neurosci Behav Physiol 36, 565–571. 25 Ikura M, Clore GM, Gronenborn AM, Zhu G, Klee CB & Bax A (1992) Solution structure of a calmodulin target peptide complex by multidimensional NMR. Science 256, 632–638. 26 Elshorst B, Hennig M, Forsterling H, Diener A, Maurer M, Schulte P, Schwalbe H, Greisinger C, Krebs J, Sch- mid H et al. (1999) NMR solution structure of a com- plex of calmodulin with a binding peptide of the Ca 2+ pump. Biochemistry 38, 12320–12332. T. Pukala et al. Cupiennin 1a from Cupiennius salei FEBS Journal 274 (2007) 1778–1784 ª 2007 The Authors Journal compilation ª 2007 FEBS 1783 27 Klee CB & Vanaman TC (1982) Calmodulin. Adv Prot Chem 35, 213–219. 28 Chin D & Means AR (2000) Calmodulin: a prototypical calcium sensor. Cell Biol 10, 322–328. 29 Hoedlich KP & Ikura M (2002) Calmodulin in action: diversity in target recognition and activation mechan- isms. Cell 108, 739–742. 30 Vetter SW & Leclerc E (2003) Novel aspects of calmo- dulin target recognition and activation. Eur J Biochem 270, 404–414. 31 Roth SM, Schmeider DM, Strobel L, van Berkum M, Means A & Wand AJ (1991) Structure of the smooth muscle myosin light-chain kinase calmodulin-binding domain peptide bound to calmodulin. Biochemistry 30, 10078–10084. 32 Meador WE, Means AR & Quiocho FA (1992) Target enzyme recognition by calmodulin: 2.4 A ˚ structure of a calmodulin peptide complex. Science 257, 1251–1255. 33 Aoyagi M, Arvai AS, Tainer JA & Getzoff ED (2003) Structural basis for endothelial nitric oxide synthase binding to calmodulin. EMBO J 22, 766–775. 34 Kataoka M, Head JF, Vorherr T, Krebs J & Carafoli E (1991) Small-angle X-ray scattering study of calmodulin bound to two peptides corresponding to parts of the calmodulin-binding domain of the plasma membrane Ca-pump. Biochemistry 30, 6247–6251. 35 Ikura M, Kay LE, Krinks M & Bax A (1991) Triple- resonance multidimensional NMR study of calmodulin complexed with the binding domain of skeletal muscle myosin light-chain kinase: indication of a conforma- tional change in the central helix. Biochemistry 30, 5498–5504. 36 Delaglio F, Grzesiek S, Vuister GW, Zhu G, Pfeifer J & Bax A (1995) NMRPipe: a multidimensional spectral processing system based on UNIX pipes. J Biomol NMR 6, 277–293. 37 Goddard TD, Kneller DG (2006). SPARKY 3. University of California, San Francisco, CA. Cupiennin 1a from Cupiennius salei T. Pukala et al. 1784 FEBS Journal 274 (2007) 1778–1784 ª 2007 The Authors Journal compilation ª 2007 FEBS . Cupiennin 1a, an antimicrobial peptide from the venom of the neotropical wandering spider Cupiennius salei, also inhibits the formation of nitric oxide. 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