10426
J. Am. Chem. SOC.1994,116, 10426-10436
Disulfide Bond Assignment of o-Agatoxins IVB and IVC: Discovery of a D-Serine Residue in o-Agatoxin IVB Steven D. Heck, Paul R. Kelbaugh, Mary E. Kelly, Peter F. Thadeio, Nicholas A. Saccomano, Justin G. Stroh, and Robert A. Volkmann' Contribution from the Department of Medicinal Chemistry and Neuroscience, Pfizer Central Research, Groton, Connecticut 06340 Received May IO, 1994@
Abstract: A TCEP (tris(2-~arboxyethyl)phosphine)-basedreductiodcysteine amidoalkylation strategy was utilized to solve the disulfide structures of w-agatoxins IVB (1) and IVC (2). These p-type calcium channel antagonists, isolated from the American funnel-web spider Agelenopsis aperta, were found to have the same amino acid sequence and disulfide bond motif. The difference between w-Aga IVB (1) and IVC (2) resides in the C-termini (Ser46) of both peptides. w-Aga IVB (1) contains a D-serine residue while w-Aga IVC (2) has an L-serine in this position. The existence of D-amino acids in eucaryotic systems is extremely rare. To our knowledge, however, this is the first time that a peptide sequence with an established cystine pattern possesses an amino acid in both D and L configurations.
Cysteine-rich proteins comprise a wide array of biologically important molecules including enzymes, protease inhibitors, plasma proteins, neurotoxins, and hormones. The disulfide bridges of these molecules play a key role in establishing and maintaining their three-dimensional structures.2 No other molecular interaction imposes such stringent structural requirements on a protein as the disulfide bond (S-S bond length of 2.05 z t 0.03 A; tortional angle about the S-S bond of -90"; angle between the disulfide bond and the P-carbon of each Cys residue of ~ 1 0 3 O ) . Disulfide ~ bonds direct the folding of individual peptide chains, while contributing to the conformational and biochemical stability of the protein Many naturally occurring toxins contain a high-density core of cysteine residues. These toxins have greatly enhanced our understanding of the pharmacology of ion channels, which are present in every cell plasma membrane and play a pivotal role in the control of a variety of physiological processes. The specificity of dendrotoxin (Dendroaspis angusticeps (mamba snake))," apamin (Apis mellifera (honey bee)),5 and charybdotoxin (Leiurus quinquestriatus var. hebraeus (scorpion))6 for K+ channel subtypes, a-scorpion toxins (Buthinae subfamily (scorpion))' for Na+ channels, and o-conotoxin GVIA (conus geographus (marine snail))* and w-agatoxin IVA (Agelenopsis uperta (funnel-web for n- and p-type Ca2+channels, respectively, has been largely responsible for the characterization of these channels. We and others are interested in voltageAbstract published in Advance ACS Abstracts, October 1 , 1994. (1) (a) Thomton, J. M. J . Mol. Biol. 1981,151,261-287. (b) Zhou, N. E.; Kay, C. M.; Hodges, R. S . Biochemistry 1993, 32, 3178-3187. (2) Creighton, T. E. BioEssays 1988, 8, 57-63. (3) Steudel, R. Angew. Chem., Int. Ed. Engl. 1975, 14, 655-664. (4) Halliwell, J. V.; Othman,I. B.; Pelchen-Matthews, A.; Dolly, J. 0. Proc. Natl. Acad. Sci. U S A . 1986, 83, 493-497. (5) Cooks, N. S.; Haylett, D. G. J. Physiol. 1985, 358, 373-394. (6) Gimenez-Gallego, G.; Navia, M. A.; Reuben, J. P.; Katz, G. M.; Kaczorowski, G. J.; Garcia, M. L. Proc. Natl. Acad. Sci. U S A . 1988, 85, 3329-3333. (7) (a) Strichartz, G.; Rando, T.; Wang, G. K. Annu. Rev. Neurosci. 1987, 10, 237-267. (b) Catterall, W. A. ISI Atlas Sci.: Phannacol. 1988, 2, 190-195. (8) (a) Aosaki, T.; Kasai, H. Pflugers Arch. 1989, 414, 150-156. (b) Rivier, J. E.; Galyean, R.; Gray, W. R.; Azimi-Zonooz, A,; McIntosh, J. M.; Cruz, L. J.; Olivera, B. M. J. Biol. Chem. 1987, 262, 1194-1198. (9) (a) Mintz, I. M.; Adams, M. E.; Bean, B. P. Neuron. 1992, 9, 8595. (b) Mintz, I. M.; Venema, V. J.; Swiderek, K. M.; Lee, T. D.; Bean, B. P.; Adams, M. E. Nature 1992, 355, 827-829. @
sensitive Ca2+ channels (VSCC) and the structure/function of w-agatoxins, which have been isolated from the American funnel-web spider Agelenopsis aperta. Our attention has focused on the p-type calcium channel antagonist w-Aga IVA9-11 in addition to two biologically similar peptides with diverging potency that we have designated w-Aga IVB (l)l2-I5 and IVC (2).15 w-Aga IVB (1) and IVC (2) have the same amino acid sequence but are distinguished chromatographically by RP-HPLC, suggesting the possibility of dissimilar disulfide bond patterns.15a Recent 2D NMR experiments12J3unambiguously identified the Cys4-Cys20 and the Cys27-Cys34 disulfide bonds of w-Aga IVB (1) and suggested the likelihood of Cys12-Cys25 and Cyslg-Cys36 disulfide bonds. Given that the last two disulfide bond assignments were not definitive and sufficient quantities of w-Aga IVC (2) were not available for a similar NMR analysis, we began to pursue alternative methods (requiring
