Delineation of the functional site of alpha-dendrotoxin. The functional topographies of dendrotoxins are different but share a conserved core with those of other Kv1 potassium channel-blocking toxins.
We identified the residues that are important for the binding of alpha-dendrotoxin (alphaDTX) to Kv1 potassium channels on rat brain synaptosomal membranes, using a mutational approach based on site-directed mutagenesis and chemical synthesis. Twenty-six of its 59 residues were individually substituted by alanine. Substitutions of Lys5 and Leu9 decreased affinity more than 1000-fold, and substitutions of Arg3, Arg4, Leu6, and Ile8 by 5-30-fold. Substitution of Lys5 by norleucine or ornithine also greatly altered the binding properties of alphaDTX. All of these analogs displayed similar circular dichroism spectra as compared with the wild-type alphaDTX, indicating that none of these substitutions affect the overall conformation of the toxin. Substitutions of Ser38 and Arg46 also reduced the affinity of the toxin but, in addition, modified its dichroic properties, suggesting that these two residues play a structural role. The other residues were excluded from the recognition site because their substitutions caused no significant affinity change. Thus, the functional site of alphaDTX includes six major binding residues, all located in its N-terminal region, with Lys5 and Leu9 being the most important. Comparison of the functional site of alphaDTX with that of DTX-K, another dendrotoxin (Smith, L. A., Reid, P. F., Wang, F. C., Parcej, D. N., Schmidt, J. J., Olson, M. A., and Dolly, J. O. (1997) Biochemistry 36, 7690-7696), reveals that they only share the predominant lysine and probably a leucine residue; the additional functional residues differ from one toxin to the other. Comparison of the functional site of alphaDTX with those of structurally unrelated potassium channel-blocking toxins from venomous invertebrates revealed the common presence of a protruding key lysine with a close important hydrophobic residue (Leu, Tyr, or Phe) and few additional residues. Therefore, irrespective of their phylogenetic origin, all of these toxins may have undergone a functional convergence. The functional site of alphaDTX is topographically unrelated to the "antiprotease site" of the structurally analogous bovine pancreatic trypsin inhibitor.
Gasparini
S
S
Département d'Ingéniérie et d'Etudes des Protéines, CEA Saclay, 91191 Gif sur Yvette Cedex, France.
Danse
J M
JM
Lecoq
A
A
Pinkasfeld
S
S
Zinn-Justin
S
S
Young
L C
LC
de Medeiros
C C
CC
Rowan
E G
EG
Harvey
A L
AL
Ménez
A
A
D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D000818:Animals; D017124:Conserved Sequence; D004546:Elapid Venoms / Q000737:chemistry / Q000378:metabolism*; D007457:Iodine Radioisotopes; D008297:Male; D008969:Molecular Sequence Data; D026902:Potassium Channel Blockers; D011485:Protein Binding; D011487:Protein Conformation; D051381:Rats; D017207:Rats, Sprague-Dawley; D017386:Sequence Homology, Amino Acid; D013574:Synaptosomes / Q000378:metabolism
D004546:Elapid Venoms; D007457:Iodine Radioisotopes; D026902:Potassium Channel Blockers; C020273:dendrotoxin