Homologous k-neurotoxins exhibit residue-specific interactions with the a3 subunit of the nicotinic acetylcholine receptor: a comparison of the structural requirements for k-bungarotoxin and k-flavitoxin binding
Mclane, K.E.; Weaver, W.R.; Lei, S.
Biochemistry (American Chemical Society) 32: 88-94
k-Flavotoxin (k-FTX), a snake neurotoxin that is a selective antagonist of certain neuronal nicotinic acetylcholine receptors (AChRs), has recently been isolated and characterized [Grant, G. A., Frazier, M. W., and Chiappinelli, V. A. (1988) Biochemistry 27, 1532-1537]. Like the related snake toxin k-bungarotoxin (k-BTX), k-FTX binds with high affinity to a3 subtypes of neuronal AChRs, even though there are distinct sequence differences between the two toxins. To further characterize the sequence regions of the neuronal AChR a3 subunit involved in formation of the binding site for this family of k-neurotoxins, we investigated k-FTX binding to overlapping synthetic peptides screening the a3 subunit sequence. A sequence region forming a "prototope" for k-FTX was identified within residues a3(51-70), confirming the suggestions of previous studies on the binding of k-BTX to the a3 subunit [McLane, K. E., Tang, F., and Conti-Tronconi, B. M. (1990) J. Biol. Chem. 265, 1537-1544] and a-bungarotoxin to the Torpedo AChR a subunit [Conti-Tronconi, B. M., Tang, F., Diethelm, B. M., Spencer, S. R., Reinhardt-Maelicke, S., and Maelicke, A. (1990) Biochemistry 29, 6221-6230] that this sequence region is involved in formation of a cholinergic site. Single residue substituted analogues, where each residue of the sequence a3(51-70) was sequentially replaced by a glycine, were used to identify the amino acid side chains involved in the interaction of this prototope with k-FTX. Substitution of several aliphatic (L54, L56, and L65), aromatic (W55, W60, W67, and Y63), and positively charged (K57, K64, K66, and K68) residues drastically reduced the ability of the peptides to interact with k-FTX. Comparison of the results of the present study with those obtained previously for the binding to the same prototope of the related neuronal toxin, k-BTX, suggests that, like the interaction of peripheral AChRs with the family of the a-neurotoxins, the interaction of neuronal AChRs with toxins of the k-neurotoxin family involves aromatic and aliphatic residues of general importance. Individual binding preference of specific toxins may involve charged amino acid residues at the toxin/AChR interface. Copyright 1993, American Chemical Society. .