CRYSTAL-STRUCTURE OF KAPPA-BUNGAROTOXIN AT 2.3-ANGSTROM RESOLUTION

kappa-Neurotoxins display a very low affinity for neuromuscular recept ors, but bind tightly to, and inhibit, nicotinic acetylcholine recepto rs in neuronal tissue such as the chick ciliary ganglia. In contrast, alpha-neurotoxins bind with high affinity and inhibit nicotinic acetyl choline receptors at the neuromuscular junction. The origin of this di fference in specificity has been a long-studied question in the field. Here we report the first crystal structure of a kappa-neurotoxin, kap pa-bungarotoxin. Unlike the NMR structure previously reported [Sutclif fe, M. J., Dobson, C. M., & Oswald, R. E. (1992) Biochemistry 31, 2962 -2970], the present crystal structure more accurately defines the poly peptide fold and the nature of the interaction between subunits in the active dimer, which is a unique feature of the kappa-neurotoxins. The structure has been refined to R = 19.6% with X-ray diffraction data e xtending to a resolution of 2.3 Angstrom. There are two independent pr otein molecules (66 amino acid residues each) in the asymmetric unit t hat are arranged as a dimer with the two subunits related by a rotatio n of 178.6 degrees Each subunit consists of three main-chain loops. Th ree of the five beta-strands of each subunit form an antiparallel beta -sheet which becomes an extended six-stranded antiparallel beta-sheet, by virtue of the approximate 2-fold symmetry of the dimer. The intera ctions at the dimer interface consist of six main-chain-main-chain hyd rogen bonds, as well as three other hydrogen-bonding interactions invo lving side chains. Residues Phe 49 and Leu 57 are found in all four ka ppa-bungarotoxins that have been sequenced, but occur in no alpha-neur otoxins, and they form van der Waals interactions across the dimer int erface. The two subunits of the dimer are not identical, with the majo r difference between them occurring at the tip of the central loop (Cy s 27-Pro 36). Residue Arg 34, which is essential for the activity of b oth alpha- and kappa-neurotoxins, occurs at the tip of the central loo p in each subunit, with guanidinium groups that are similar to 44 Angs trom apart.