Site-directed mutagenesis of dendrotoxin K reveals amino acids critical for its interaction with neuronal K+ channels.
Dendrotoxin K (DTXK) is a 57-residue protein from mamba venom that blocks certain non-inactivating, voltage-activated K+ currents in neurones. In order to pinpoint the residues responsible for its specificity, structure-activity relations of DTX(K) were investigated by mutagenesis. A previously cloned gene encoding this toxin [Smith et al. (1993) Biochemistry 32, 5692-5697] was used to make single mutations; after expression in Escherichia coli as fusion proteins and enzymatic cleavage of the conjugates isolated from the periplasmic space, nine toxins were purified. Structural analysis of the native DTXK and representative mutants by circular dichroism showed that no significant differences were detectable in their folded structures. The biological activity of the mutants, which contained alterations of positively charged and other amino acids, was determined from their abilities to compete for the binding of 125I-labeled DTX(K) to K+ channels in synaptic plasma membranes from rat cerebral cortex. Mutants with residues substituted in the alpha-helix near the C-terminus (R52A or R53A) yielded binding parameters similar to those of wild-type and native DTX(K). In the case of the beta-turn (residues 24-28), however, altering single amino acids reduced binding to the high-affinity site of K+ channels, with the rank order of decreases being K26A >> W25A > K24A = K28A. Also, substitutions made in the 3(10)-helix (residues 3-7), a region located close to the beta-turn, produced equivalent effects (K3A > K6A). Thus, it is deduced that residues in the distorted beta-turn and neighboring 3(10)-helix of DTX(K) are critical for its interaction with neuronal K+ channels.
Smith
L A
LA
Department of Immunology and Molecular Biology, United States Army Medical Research Institute of Infectious Diseases, Fort Detrick, Frederick, Maryland 21702-5011, USA.
Reid
P F
PF
Wang
F C
FC
Parcej
D N
DN
Schmidt
J J
JJ
Olson
M A
MA
Dolly
J O
JO
D000818:Animals; D004546:Elapid Venoms / Q000737:chemistry*; D016297:Mutagenesis, Site-Directed; D009474:Neurons / Q000378:metabolism*; D010455:Peptides / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D015221:Potassium Channels / Q000378:metabolism*; D011485:Protein Binding; D051381:Rats; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism
D004546:Elapid Venoms; D010455:Peptides; D015221:Potassium Channels; D011994:Recombinant Proteins; C081506:dendrotoxin K