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• W2080486908 abstract "Mechanosensitive channels (MSCs) play key roles in sensory processing and have been implicated as primary transducers for a variety of cellular responses ranging from osmosensing to gene expression. This paper presents the first structures of any kind known to interact specifically with MSCs. GsMTx-4 and GsMtx-2 are inhibitor cysteine knot peptides isolated from venom of the tarantula,Grammostola spatulata (Suchyna, T. M., Johnson, J. H., Hamer, K., Leykam, J. F., Gage, D. A., Clemo, H. F., Baumgarten, C. M., and Sachs, F. (2000) J. Gen. Physiol. 115, 583–598). Inhibition of cationic MSCs by the higher affinity GsMtx-4 (KD ∼500 nm) reduced cell size in swollen and hypertrophic heart cells, swelling-activated currents in astrocytes, and stretch-induced arrhythmias in the heart. Despite the relatively low affinity, no cross-reactivity has been found with other channels. Using two-dimensional NMR spectroscopy, we determined the solution structure of GsMTx-4 and a lower affinity (GsMTx-2; KD ∼6 μm) peptide from the same venom. The dominant feature of the two structures is a hydrophobic patch, utilizing most of the aromatic residues and surrounded with charged residues. The spatial arrangement of charged residues that are unique to GsMTx-4 and GsMTx-2 may underlie the selectivity of these peptides. Mechanosensitive channels (MSCs) play key roles in sensory processing and have been implicated as primary transducers for a variety of cellular responses ranging from osmosensing to gene expression. This paper presents the first structures of any kind known to interact specifically with MSCs. GsMTx-4 and GsMtx-2 are inhibitor cysteine knot peptides isolated from venom of the tarantula,Grammostola spatulata (Suchyna, T. M., Johnson, J. H., Hamer, K., Leykam, J. F., Gage, D. A., Clemo, H. F., Baumgarten, C. M., and Sachs, F. (2000) J. Gen. Physiol. 115, 583–598). Inhibition of cationic MSCs by the higher affinity GsMtx-4 (KD ∼500 nm) reduced cell size in swollen and hypertrophic heart cells, swelling-activated currents in astrocytes, and stretch-induced arrhythmias in the heart. Despite the relatively low affinity, no cross-reactivity has been found with other channels. Using two-dimensional NMR spectroscopy, we determined the solution structure of GsMTx-4 and a lower affinity (GsMTx-2; KD ∼6 μm) peptide from the same venom. The dominant feature of the two structures is a hydrophobic patch, utilizing most of the aromatic residues and surrounded with charged residues. The spatial arrangement of charged residues that are unique to GsMTx-4 and GsMTx-2 may underlie the selectivity of these peptides. mechanosensitive channel correlation spectroscopy mechanotoxins from G. spatulata venom heteronuclear single quantum correlation inhibitor cysteine knot nuclear Overhauser effect NOE spectroscopy stretch-activated channel total correlation spectroscopy high pressure liquid chromatography root mean square 3-bromo-3-methyl-2[(2-nitrophenyl)thiol]-3H-indole All cells are responsive to mechanical stimulation, and mechanosensitive ion channels (MSCs)1 are the most sensitive transducers (1Sachs F. Morris C.E. Blaustein M.P. Greger R. Grunicke H. Jahn R. Mendell L.M. Miyajima A. Pette D. Schultz G. Schweiger M. Reviews of Physiology, Biochemistry, and Pharmacology. Springer, Berlin1998: 1-78Google Scholar). In higher animals, exteroceptorstransduce external stimuli such as sound, vibration, touch, and local gravity, whereas interoceptors provide feedback for the voluntary musculature and autonomic signals that provide information on filling of the hollow organs (blood pressure regulation, filling of the bladder, etc.). Interoceptors include cellular transducers that permit local control of blood flow, dilation-induced changes in heart rate, and regulation of cell volume and thirst; hormonal-coupled transducers that convert mechanical stimuli into release of factors such as renin and atrial naturietic factor; and autocrine and paracrine transducers that are responsible for local secretion of agents such as endothelin and other growth factors and gene regulators that affect cell division and cell size, as in bone and muscle growth. MSCs are the only known primary mechanical transducers and may drive many of these processes. The three-dimensional structure of a bacterial MSC is known (2Chang G. Spencer R.H. Lee A.T. Barclay M.T. Rees D.C. Science. 1998; 282: 2220-2226Crossref PubMed Scopus (863) Google Scholar), but in eukaryotes, the only MSCs that have been cloned are a family of K+-selective channels (3Patel A.J. Honore E. Maingret F. Lesage F. Fink M. Duprat F. Lazdunski M. EMBO J. 1998; 17: 4283-4290Crossref PubMed Scopus (537) Google Scholar, 4Patel A.J. Honore E. Trends Neurosci. 2001; 24: 339-346Abstract Full Text Full Text PDF PubMed Scopus (377) Google Scholar, 5Patel A.J. Lazdunski M. Honore E. Curr. Opin. Cell Biol. 2001; 13: 422-428Crossref PubMed Scopus (246) Google Scholar). The number of different subtypes of MSCs in eukaryotes is unknown. They can be classified in a broad spectrum as stretch-activated channels (SACs) and stretch-inactivated channels. Some of each are K+-selective, and others are nonselective cation channels (6Hamill O.P. Martinac B. Physiol. Rev. 2001; 81: 685-740Crossref PubMed Scopus (930) Google Scholar). Despite the widespread nature of mechanical transduction in biology, no specific activator or inhibitors of MSCs have been known until recently. Gd3+ (10–20 μm) inhibits SACs in both plant and animal cells, suggesting a functional homology between the channels, but Gd3+ is not specific for SACs; nor can it be considered a useful lead compound for the development of therapeutic agents. Furthermore, it is not clear if the effects of Gd3+are directly on SACs or mediated by a stiffening of membrane lipids (7Ermakov Y.A. Averbakh A.Z. Yusipovich A.I. Sukharev S. Biophys. J. 2001; 80: 1851-1862Abstract Full Text Full Text PDF PubMed Scopus (86) Google Scholar, 8Ermakov Yu A. Averbakh A.Z. Arbuzova A.B. Sukharev S.I. Membr. Cell Biol. 1998; 12: 411-426PubMed Google Scholar, 9Ermakov Yu A. Averbakh A.Z. Sukharev S.I. Membr. Cell Biol. 1997; 11: 539-554PubMed Google Scholar). Both amiloride (10Hackney C.M. Furness D.N. Am. J. Physiol. 1995; 268: C1-C13Crossref PubMed Google Scholar, 11Hamill O.P. McBride D.W., Jr. Pharmacol. Rev. 1996; 48: 231-252PubMed Google Scholar, 12Rusch A. Kros C.J. Richardson G.P. J. Physiol. 1994; 474: 75-86Crossref PubMed Scopus (110) Google Scholar, 13Small D.L. Morris C.E. Br. J. Pharmacol. 1995; 114: 180-186Crossref PubMed Scopus (27) Google Scholar) and some cationic antibiotics (11Hamill O.P. McBride D.W., Jr. Pharmacol. Rev. 1996; 48: 231-252PubMed Google Scholar) can inhibit SACs, but again they lack specificity. Recently, we discovered a family of peptides from the venom of the Chilean rose tarantula,Grammostola spatulata, that inhibit SACs in astrocytes and heart cells. The more potent of these toxins, GsMTx-4, not only blocks MSC currents seen with patch clamp recording but exhibits effects at the cellular level that appear to involve MSC activation. GsMTx-4 can reduce the cell size in swollen and hypertrophic heart cells, decrease volume-activated currents in astrocytes (14Suchyna T.M. Johnson J.H. Hamer K. Leykam J.F. Gage D.A. Clemo H.F. Baumgarten C.M. Sachs F. J. Gen. Physiol. 2000; 115: 583-598Crossref PubMed Scopus (265) Google Scholar), and inhibit stretch-induced arrhythmias in the heart (15Bode F. Sachs F. Franz M.R. Nature. 2001; 409: 35-36Crossref PubMed Scopus (266) Google Scholar). We have determined the solution structure of GsMTx-4 and a lower affinity inhibitor (GsMTx-2) using homonuclear and heteronuclear two-dimensional NMR spectroscopy. The backbone folds of both peptides exhibit the inhibitor cysteine knot (ICK) motif, which is characteristic of a family of invertebrate toxins directed toward a variety of ion channels (16Craik D.J. Daly N.L. Waine C. Toxicon. 2001; 39: 43-60Crossref PubMed Scopus (418) Google Scholar, 17Norton R.S. Pallaghy P.K. Toxicon. 1998; 36: 1573-1583Crossref PubMed Scopus (276) Google Scholar) as well as at least one mammalian protein (18McNulty J.C. Thompson D.A. Bolin K.A. Wilken J. Barsh G.S. Millhauser G.L. Biochemistry. 2001; 40: 15520-15527Crossref PubMed Scopus (72) Google Scholar). A dominant feature of the structures is a hydrophobic patch, similar to that described for Hanatoxin (19Takahashi H. Kim J.I. Min H.J. Sato K. Swartz K.J. Shimada I. J. Mol. Biol. 2000; 297: 771-780Crossref PubMed Scopus (131) Google Scholar), encompassing most of the aromatic residues that have been suggested to be important for toxin-receptor interaction. As with Hanatoxin, MSC peptides have charged residues surrounding the hydrophobic patch, but the distribution of the charges differs between GsMTx-4 and GsMTx-2, suggesting structural correlates that may account for differences in affinity. Hanatoxin is ineffective on MSCs. The ICK motif can be viewed as a well defined scaffold upon which side chains are placed to produce specificity for a particular target. Knowing the structure of MSC-directed ICK peptides, we can design modified structures that are labeled with various indicators, peptides that have higher affinity, and peptides that have differing specificity within the family of MSCs. A high affinity tag may allow isolation of this very difficult group of ion channels. The method for venom fractionation and toxin isolation is described in detail by Suchyna et al.(14Suchyna T.M. Johnson J.H. Hamer K. Leykam J.F. Gage D.A. Clemo H.F. Baumgarten C.M. Sachs F. J. Gen. Physiol. 2000; 115: 583-598Crossref PubMed Scopus (265) Google Scholar). Briefly, G. spatulata (Theraphosidae) spider venom was produced by an electrical milking procedure (20Bascur L. Yevenes I. Adrian H. Toxicon. 1980; 18: 224Crossref PubMed Scopus (12) Google Scholar) and stored at −80 °C. The venom was fractionated by high performance liquid chromatography, incorporating Beckman System Gold 126 solvent delivery and 168 photodiode array detector modules (Beckman Instruments, Fullerton, CA) using linear gradients of solvent A (0.1% trifluoroacetic acid in water) and solvent B (0.1% trifluoroacetic acid in acetonitrile). A Zorbax RX-C8 (9.4 × 250 mm, 5 μm, 300 Å; Mac-Mod Analytical, Inc., Chadds Ford, PA) reversed-phase column was used in conjunction with a 40-min linear gradient (15–55% B) with a flow rate of 3.5 ml/min. Fractions were lyophilized and tested for MSC blocking activity on outside-out patches. Fractions that tested positive were further fractionated with slower gradients on a Vydac C18 column (10 × 250 mm, 5 μm, 300 Å; The Separations Group, Hesperia, CA) until a single component was isolated that showed blocking activity. The purity of specific peaks was assessed by analytical chromatography on a Jupiter C18 column (4.6 × 150 mm, 5 μm, 300 Å; Phenomenex, Torrance, CA) with a 40-min gradient of 20–28% B. The average yield of GsMTx-4 and GsMTx-2 from several purifications was ∼8 and ∼24 mg/ml of venom fractionated, respectively. Thus, GsMTx-4 is at least 2 mmin whole venom, whereas GsMTx-2 is at least 6 mm. 0.5 μl of the sample solution in 0.1% trifluoroacetic acid was mixed on the sample plate with 0.5 μl of a saturated solution of 4-hydroxy-α-cyanocinnaminic acid in 50% acetonitrile, 0.1% trifluoroacetic acid containing insulin as the internal calibrant. The solution was air-dried before introduction into the mass spectrometer. Spectra were acquired on a PerSeptive Voyager-DE STR matrix-assisted laser desorption ionization time-of-flight instrument operated in reflectron delayed extraction mode (200 ns). The instrument was equipped with a nitrogen laser (3-ns pulse, 1950 power). The accelerating potential was 25 kV, and the grid voltage was 72%. The toxin was further purified by microbore reversed-phase HPLC (0.8 × 250-mm C18 column, with a linear gradient from 15–70% B over 90 min, with a flow rate of 40 μl/min, monitored at 214 nm). The toxin peak was collected at 24.6 min. The HPLC fraction (∼1 nmol) was dried down and taken up in 80 μl of 8m guanidine-HCl, 100 mm Tris, 5 mmtributylphosphine at pH 8.5 and incubated for 8 h at 55 °C.N-Isopropyliodoactamide (1 mg in 20 μl of MeOH plus 80 μl of Tris) was added, and the solution was incubated for an additional 2 h at room temperature. The reduced and alkylated peptide was then desalted by HPLC on a C18 column as described above (elution time 30.1 min). N-terminal sequencing was carried out on an ABI 477 after loading the reduced and alkylated peptide on polyvinylidene difluoride membrane. Digestion with BNPS-skatole was carried out by dissolving the purified reduced and alkylated peptide in 50 μl of 0.1% trifluoroacetic acid and 15 μl of BNPS-skatole. The solution was incubated at room temperature for 8 h. The digestion products were separated by HPLC as described above. Two main peaks were collected and sequenced by Edman degradation. Asp-N digestion was performed by dissolving the purified reduced and alkylated peptide in 100 mm Tris, pH 8.0, and treating with 1% (w/w) Asp-N for 20 h at 35 °C. The fragments were separated and analyzed by mass spectrometry prior to Edman degradation. For mass spectrometry, 1 μl of the sample solutions (intact toxin or fragments) in 0.1% trifluoroacetic acid (or the HPLC elution solvent) were mixed on the sample plate with 1 μl of a saturated solution of 4-hydroxy-α-cyanocinnamic acid in 50% acetonitrile, 0.1% trifluoroacetic acid. The solution was allowed to air-dry before being introduced into the mass spectrometer. Activated adult astrocytes isolated from gelatin-sponge implants taken from adult Sprague-Dawley rat brain were generously provided by Dr. Thomas Langan (SUNY Buffalo) at passage numbers 2–4. Astrocytes were maintained in Dulbecco’s modified Eagle’s medium, 10% fetal bovine serum, and 1% penicillin/streptomycin and were used in experiments between 2 and 5 days after passage. Cells between passages 4 and 35 expressed SACs with the same properties. Flat, polygonal, fibroblast-like cells and stellate cells were used. An Axopatch 200B (Axon Instruments) amplifier was used for patch clamping, whereas experimental protocols and data acquisition were controlled by Axon Instruments pClamp8 software via a Digidata 1322A acquisition system. Currents were sampled at 10 kHz and low pass-filtered at 2 kHz through the four-pole Bessel filter on the Axopatch 200B. All potentials are defined with respect to the extracellular surface. Electrodes were pulled on a model PC-84 pipette puller (Brown-Flaming Instruments, CA), painted with Sylgard 184 (Dow Corning Corp., Midland, MI) and fire-polished. Electrodes were filled with KCl saline (140 mm KCl, 5 mm EGTA, 2 mmMgSO4, 10 mm Hepes, pH 7.3) and had resistances ranging from 6 to 8 megaohms. Bath saline consisted of 140 mm NaCl, 5 mm KCl, 2 mmCaCl2, 0.5 mm MgSO4, 6 mm glucose, and 10 mm Hepes, pH 7.3. Pressure and suction were applied to the pipette by a HSPC-1 pressure clamp (ALA Scientific Instruments) controlled by the pClamp software. Perfusion of toxin samples was performed by a pressurized local perfusion system BPS-8 (ALA Scientific Instruments) with eight separate channels. Off-line data analysis was performed with Clampfit and Origin 6.1 software. In order to determine whether the proteins were aggregated, the coefficients of self-diffusion (Dt) for GsMTx2 and GsMTx4 were measured using a longitudinal encode-decode pulse sequence (21Altieri A.S. Hinton D.P. Byrd R.A. J. Am. Chem. Soc. 1995; 117: 7566-7567Crossref Scopus (441) Google Scholar) with the proteins lyophilized and resuspended in D2O. Lysozyme was used as a standard control for slope calibration. Dt for 2 mm lysozyme in a1H2O/2H2O mixture at 298 K was taken to be 1.08 × 10−6 cm2s−1 (21Altieri A.S. Hinton D.P. Byrd R.A. J. Am. Chem. Soc. 1995; 117: 7566-7567Crossref Scopus (441) Google Scholar). Data from experiments utilizing 10 different gradient strengths, ranging from 4 to 60 gauss, were analyzed. Increments of 1/8, 1/4, ½, and 3/4 maximum were run in triplicate to provide estimates of error in the exponential fit. Peak intensities were fit to the equation,ln(I)=ln(I0)+G2γ2δ2(Δ/3−δ/3)DtEquation 1 where I represents measured peak intensity,G is the gradient strength in gauss/cm, γ is the gyromagnetic ratio of protons (26,753 radian s−1G−1), δ is the length of the gradient pulse (1.5 ms), Δ is the diffusion time between gradients (600 ms), andDt is the self-diffusion rate. Samples from the Vydac C18 column were lyophilized and resuspended in distilled H2O to 2 mm toxin. The samples were then titrated to pH 4.5, and D2O was added to 8%. A 250-μl aliquot of the protein solution was transferred to a 5-mm Shigemi tube for NMR spectroscopy. All experiments were performed on Varian Inova 600 and 500 spectrometers equipped with triple resonance Z-gradient probes at the Cornell Biomolecular NMR Center. Data were collected in States-TPPI mode (22States D.J. Haberkorn R.A. Ruben D.J. J. Magn. Reson. 1982; 48: 286-292Google Scholar) for quadrature detection. Homonuclear two-dimensional NOESY (23Kumar A. Ernst R.R. Wu¨thrich K. Biochem. Biophys. Res. Commun. 1980; 95: 1-6Crossref PubMed Scopus (2031) Google Scholar), TOCSY (24Braunschweiler L. Ernst R.R. J. Mag. Res. 1983; 53: 521-528Crossref Scopus (3111) Google Scholar), and COSY (25Aue W.P. Bartholdi E. Ernst R.R. J. Chem. Phys. 1976; 64: 2229-2246Crossref Scopus (3098) Google Scholar) spectra were obtained at 5, 16, 20, 25, and 35 °C with presaturation during the recycle period followed by SCUBA recovery (26Brown S.C. Weber P.L. Mueller L. J. Mag. Res. 1988; 77: 166-169Google Scholar). A DIPSI-2 (27Rucker S.P. Shaka A.J. Mol. Phys. 1989; 68: 509-517Crossref Scopus (269) Google Scholar) sequence of 60–90 ms was used in the TOCSY experiments, and a 150-ms mixing period was used in the NOESY experiments. Two-dimensional 1H,15N HSQC and1H,13C HSQC (28Bodenhausen G. Ruben D.J. Chem. Phys. Lett. 1980; 69: 185-189Crossref Scopus (2435) Google Scholar) spectra were acquired with natural abundance proteins. Data were processed either with a modification of version 2.3 of Felix software (Accelrys, Inc.) or NMRPipe (29Delaglio F. Grzesiek S. Vuister G. Zhu G. Pfeifer J. Bax A. J. Biomol. NMR. 1995; 6: 277-293Crossref PubMed Scopus (11632) Google Scholar). Sparky (30Goddard, T. D., and Kneller, D. G., Sparky, Version 3, University of California, San FranciscoGoogle Scholar) was used for data visualization, assignments, and peak integration. Dihedral angle constraints were obtained from the chemical shift index (31Wishart D.S. Sykes B.D. Richards R.M. J. Mol. Biol. 1991; 222: 311-333Crossref PubMed Scopus (1791) Google Scholar, 32Wishart D.S. Sykes B.D. Richards F.M. Biochemistry. 1992; 31: 1647-1651Crossref PubMed Scopus (2024) Google Scholar, 33Wishart D.S. Sykes B.D. J. Biomol. NMR. 1994; 4: 171-180Crossref PubMed Scopus (1916) Google Scholar) and from measurements of coupling constants using a two-dimensional COSY experiment with 8192 points in t2. Stereospecific assignments and χ1 constraints were determined using an exclusive COSY (34Griesinger C. Sorensen O.W. Ernst R.R. J. Magn. Res. 1987; 75: 747-792Google Scholar) and the NOESY experiments. Distance constraints for structural calculations were obtained from a series of two-dimensional homonuclear NOESY experiments at 5–35 °C in either 90% H2O/10% D2O or 100% D2O. Distance constraints were classified into four categories according to the intensity of the NOE cross-peak (<2.4, <3.4, <4.0, and <5.5 Å). On the basis of the chemical shift index and coupling constants from a high resolution COSY experiment, backbone dihedral angles were constrained to favorable regions of φ,ψ space: α-helix, φ, −80 ± 30°; ψ, −20 ± 30°; β-strand, φ, −105 ± 65°; ψ, 145 ± 45° (35Laskowski R.A. Rullmannn J.A. MacArthur M.W. Kaptein R. Thornton J.M. J. Biomol. NMR. 1996; 8: 477-486Crossref PubMed Scopus (4470) Google Scholar). χ1 constraints and stereospecific assignments of β protons were made based on the exclusive COSY and NOESY spectra (36Wagner G. Braun W. Havel T.F. Schaumann T., Go, N. Wu¨thrich K. J. Mol. Biol. 1987; 196: 611-639Crossref PubMed Scopus (635) Google Scholar). The distance and dihedral restraints were used as inputs to determine preliminary structures using the distance geometry/simulated annealing protocol in CNS 1.0 (Crystallography and NMR System) (37Brunger A.T. Adams P.D. Clore G.M. DeLano W.L. Gros P. Grosse-Kunstleve R.W. Jiang J.S. Kuszewski J. Nilges M. Pannu N.S. Read R.J. Rice L.M. Simonson T. Warren G.L. Acta Crystallogr. Sect. D Biol. Crystallogr. 1998; 54: 905-921Crossref PubMed Scopus (16979) Google Scholar). Hydrogen bonding constraints were added in subsequent calculations based on deuterium exchange data, characteristic NOE patterns (see Fig. 3), and likely hydrogen bonding partners determined from the preliminary structures. Three hundred structures were calculated using CNS, and the 20 lowest energy structures were aligned to the average structure using XPLOR version 3.851 (38Brunger A.T. Xplor Manual: Version 3.843. Yale University, 1996Google Scholar). The structures were visualized using Swiss-PdbView (39Guex N. Peitsch M.C. Electrophoresis. 1997; 18: 2714-2723Crossref PubMed Scopus (9640) Google Scholar) and analyzed using Procheck-NMR, Aqua (35Laskowski R.A. Rullmannn J.A. MacArthur M.W. Kaptein R. Thornton J.M. J. Biomol. NMR. 1996; 8: 477-486Crossref PubMed Scopus (4470) Google Scholar), and NMRCLUST (40Kelley L.A. Gardner S.P. Sutcliffe M.J. Protein Eng. 1996; 9: 1063-1065Crossref PubMed Scopus (419) Google Scholar). The hydrogen-bonding pattern was verified by recalculating a set of 200 structures without the hydrogen bonding constraints. The 20 lowest energy structures were analyzed. The NH and carbonyl oxygens of the hydrogen bonding pairs were oriented correctly in almost all structures, although in some cases, the distance between the NH and oxygen was somewhat greater than that observed when the hydrogen bonding constraints were included in the calculation. G. spatulatawhole venom was fractionated by reversed-phase HPLC, and fractions were superfused onto outside-out patches from rat astrocytes to assess MSC activity. Fractions that showed activity were again fractionated on successively shallower gradients until a single active component was isolated. Suchyna et al. (14Suchyna T.M. Johnson J.H. Hamer K. Leykam J.F. Gage D.A. Clemo H.F. Baumgarten C.M. Sachs F. J. Gen. Physiol. 2000; 115: 583-598Crossref PubMed Scopus (265) Google Scholar) described the properties and primary structure of GsMTx-4, a peptide with the highest affinity of any known blocker of MSCs. At a concentration of 5 μm, GsMTx-4 blocked ∼92% of the MSC current with an association rate of 3.4 × 105m−1s−1 (Fig. 1A). The ratio of association and dissociation rates produced an equilibrium constant of ∼500 nm. A similar number resulted from treating the reaction as a simple bimolecular reaction and using the residual (steady state) current as a measure of the unbound fraction (see Figs. 5 and 6 in Suchyna et al. (14Suchyna T.M. Johnson J.H. Hamer K. Leykam J.F. Gage D.A. Clemo H.F. Baumgarten C.M. Sachs F. J. Gen. Physiol. 2000; 115: 583-598Crossref PubMed Scopus (265) Google Scholar)). The largest HPLC peak in Grammostola whole venom was actually a complex of several peaks, one of which could block MSCs but showed lower activity than GsMtx-4. The active peak eluted at 21.5 min on a 15–50% acetonitrile 40-min linear gradient (see Fig. 3A in Ref. 14Suchyna T.M. Johnson J.H. Hamer K. Leykam J.F. Gage D.A. Clemo H.F. Baumgarten C.M. Sachs F. J. Gen. Physiol. 2000; 115: 583-598Crossref PubMed Scopus (265) Google Scholar). At 5 μm, this peptide (designated GsMTx-2) blocks ∼45% of the MSC current with an association rate constant of 1.7 × 105m−1 s−1 (Fig. 1B). Assuming a single binding site, the ratio of the residual unblocked steady state current (I) to the peak current in the absence of toxin (Io) is I/Io= 1/(1 + T/KD), where T is the toxin concentration. For 5 μm GsMTx-2,I/Io is 0.55, giving aKD of ∼6 μm. Thus, the affinity of GsMTx-2 for astrocyte MSCs is ∼12 times lower than that of GsMTx-4. The sequence of GsMTx-2 has less than 25% homology to GsMTx-4 as shown at thebottom of the sequence alignment in Fig. 1C. The four ICK tarantula peptides shown in the middle of Fig.1C have the highest sequence similarity to GsMTx-4 (and higher than that of GsMTx-2) but are not mechanotoxins (toxins that inhibit MSCs). Given that the peptides that are not mechanotoxins have much higher sequence similarity to GsMTx-4 than GsMTx-2, the structures of GsMTx-2 and GsMTx-4 may provide clues as to the origin of specificity for MSCs. Of the peptide sequences shown, three-dimensional structures only exist for Hanatoxin, GsMTx-2, and GsMTx-4 (see below). For these three peptides, the hydrophobic amino acids that form a cluster on the surface of the three peptides are shown in green (Fig. 1). Assuming structural homology, the corresponding hydrophobic amino acids on the other three toxins (TXP5, SNX-482, and ω-GsTx-S1A) are also shown in green. The hydrophobic patch of Hanatoxin has been suggested to be part of the channel-binding domain, based on homology to other toxins. The charged residues that surround the hydrophobic patch are shown in blue (+) and lavender (−), but since their sequence positions vary significantly, the charges are only marked on the toxins where structures are known. In order to determine whether GsMTx2 and GsMTx4 were aggregated in the samples used for NMR spectroscopy,Dt was measured at 500 MHz using a pulsed field gradient method (21Altieri A.S. Hinton D.P. Byrd R.A. J. Am. Chem. Soc. 1995; 117: 7566-7567Crossref Scopus (441) Google Scholar) with lysozyme as a standard. Plots of ln(I) versus G2 (Equation 1) were linear over the range of gradient strengths used for experiments for all three proteins (the two toxins and lysozyme). The averageDt for GsMTx2 was 2.26 × 10−6cm2 s−1, and that for GsMTx4 was 2.22 × 10−6 cm2 s−1. Using the known hydrodynamic radius of lysozyme (Rh = 20.5 Å),Rh of GsMTx2 and GsMTx4 could be estimated as ∼12.8 and 13.3 Å, respectively (41Wilkins D.K. Grimshaw S.B. Receveur V. Dobson C.M. Jones J.A. Smith L.J. Biochemistry. 1999; 38: 16424-16431Crossref PubMed Scopus (830) Google Scholar). These values are what would be expected given the empirical relationship, Rh = 4.75n0.29, for the hydrodynamic radius of a globular protein having n = 31 and 35 amino acids (41Wilkins D.K. Grimshaw S.B. Receveur V. Dobson C.M. Jones J.A. Smith L.J. Biochemistry. 1999; 38: 16424-16431Crossref PubMed Scopus (830) Google Scholar). Likewise, these values are consistent with the overall shape and dimensions of the calculated structures described below, confirming that, under the conditions used, both toxins are monomeric. Sequence-specific resonance assignments (excluding the first residue) were made for both peptides using both homonuclear and heteronuclear experiments. A natural abundance 1H,15N HSQC experiment provided a fingerprint of the amide nitrogen and amide proton correlation for each residue except proline (Fig.2). Homonuclear TOCSY, DQF-COSY, and NOESY experiments were then used to do the bulk of the assignments, which employed standard techniques (42Wu¨thrich K. NMR of Proteins and Nucleic Acids. John Wiley & Sons, Inc., New York1986Crossref Google Scholar). The assignments were assisted by the analysis of a 1H,13C HSQC spectrum. Using the proton chemical shifts in this spectrum, the peaks could be assigned to a particular spin system by comparison with the TOCSY experiment. The corresponding carbon chemical shift of the Cα and Cβ could then be used to aid in identification of the amino acid type. Backbone NOEs used to make sequential assignments and to aid in the assignment of hydrogen bonds are summarized in Fig. 3A. Using chemical shift index analysis and analysis of the high resolution DQF-COSY spectra, a total of 14 (GsMTx-4) and 17 (GsMTx-2) φ,ψ distance constraints were obtained. Characteristic of the ICK motif, both GsMTx-2 and GsMTx-4 exhibit three disulfide bonds in relatively close proximity. For GsMTx-4, if the structures are calculated without assuming a disulfide-bonding pattern, the bond between Cys2 and Cys17 can clearly be assigned due to the proximity of the two side chains and an NOE between an Hβ of Cys2 and an Hβ of Cys17. Of the remaining four cysteines, Cys9 is positioned close to both Cys30 and Cys23(3.69 ± 0.32 Å and 4.13 ± 0.69 Å, respectively). If one assumes a disulfide bond between Cys9 and Cys30, then Cys16 and Cys23 would not be positioned correctly to form a disulfide (9.51 ± 0.99 Å apart). On the other hand, if Cys9 formed a disulfide with Cys23, then Cys30 could easily form a disulfide with Cys16. This pattern is supported by an observed NOE between an Hβ of Cys16 and an Hβ of Cys30. The lowest energy structures and lowest r.m.s. deviations were obtained from structures with explicit disulfides between Cys9–Cys23 and Cys16–Cys30 as opposed to the two other possible patterns (Cys9–Cys30/Cys16–Cys23and Cys9–Cys16/Cys23–Cys30). In order to test this pattern further, structures were calculated, restraining the disulfide dihedral to within ±10° of the ideal values (i.e. 90 ± 10° and −90 ± 10°). 50 structures were calculated for each of three combinations of disulfide bonds holding 2–17 constant (Cys9–Cys23/Cys16–Cys30, Cys9–Cys30/Cys16–Cys23, and Cys9–Cys16/Cys23–Cys30) and each of eight combinations of two dihedral constraints (90 ± 10° and −90 ± 10°) on the three disulfides (i.e.400 structures for each of the three disulfide-bonding patterns). For Cys2–Cys17/Cys9–Cys30/Cys16–Cys23, less than 3% of the structures had no NOE violations, and all had relatively high overall energies. Both Cys2–Cys17/Cys9–Cys23/Cys16-Cys30and" @default.
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• W2080486908 date "2002-09-01" @default.
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• W2080486908 title "Solution Structure of Peptide Toxins That Block Mechanosensitive Ion Channels" @default.
• W2080486908 cites W1499222776 @default.
• W2080486908 cites W1518078436 @default.
• W2080486908 cites W1548025383 @default.
• W2080486908 cites W1757122036 @default.
• W2080486908 cites W1966781760 @default.
• W2080486908 cites W1975459052 @default.
• W2080486908 cites W1976595458 @default.
• W2080486908 cites W1986394529 @default.
• W2080486908 cites W1995017064 @default.
• W2080486908 cites W1998295767 @default.
• W2080486908 cites W2005008560 @default.
• W2080486908 cites W2006731694 @default.
• W2080486908 cites W2008386999 @default.
• W2080486908 cites W2008890525 @default.
• W2080486908 cites W2010528272 @default.
• W2080486908 cites W2015642465 @default.
• W2080486908 cites W2019390598 @default.
• W2080486908 cites W2020872723 @default.
• W2080486908 cites W2028231353 @default.
• W2080486908 cites W2040253039 @default.
• W2080486908 cites W2044995040 @default.
• W2080486908 cites W2045446885 @default.
• W2080486908 cites W2045950270 @default.
• W2080486908 cites W2056961349 @default.
• W2080486908 cites W2057350812 @default.
• W2080486908 cites W2061761037 @default.
• W2080486908 cites W2069734146 @default.
• W2080486908 cites W2075420089 @default.
• W2080486908 cites W2078248419 @default.
• W2080486908 cites W2085255443 @default.
• W2080486908 cites W2085451289 @default.
• W2080486908 cites W2088848851 @default.
• W2080486908 cites W2089618587 @default.
• W2080486908 cites W2089894835 @default.
• W2080486908 cites W2090215761 @default.
• W2080486908 cites W2102978118 @default.
• W2080486908 cites W2111771803 @default.
• W2080486908 cites W2116294708 @default.
• W2080486908 cites W2147381068 @default.
• W2080486908 cites W2147869016 @default.
• W2080486908 cites W2148521301 @default.
• W2080486908 cites W2150237382 @default.
• W2080486908 cites W2157194890 @default.
• W2080486908 cites W2161672145 @default.
• W2080486908 cites W2169821755 @default.
• W2080486908 cites W2170096428 @default.
• W2080486908 cites W2337927817 @default.
• W2080486908 cites W4251926840 @default.
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