The three-dimensional structural surface of two beta-sheet scorpion toxins mimics that of an alpha-helical dihydropyridine receptor segment

Abstract

An alpha-helical II-III loop segment of the dihydropyridine receptor activates the ryanodine receptor calcium-release channel. We describe a novel manipulation in which this agonist's activity is increased by modifying its surface structure to resemble that of a toxin molecule. In a unique system, native beta-sheet scorpion toxins have been reported to activate skeletal muscle ryanodine receptor calcium channels with high affinity by binding to the same site as the lower-affinity alpha-helical dihydropyridine receptor segment. We increased the alignment of basic residues in the alpha-helical peptide to mimic the spatial orientation of active residues in the scorpion toxin, with a consequent 2-20-fold increase in the activity of the alpha-helical peptide. We hypothesized that, like the native peptide, the modified peptide and the scorpion toxin may bind to a common site. This was supported by (i) similar changes in ryanodine receptor channel gating induced by the native or modified alpha-helical peptide and the beta-sheet toxin, a 10-100-fold reduction in channel closed time, with a < or = 2-fold increase in open dwell time and (ii) a failure of the toxin to further activate channels activated by the peptides. These results suggest that diverse structural scaffolds can present similar conformational surface properties to target common receptor sites.

Functional implications of modifying RyR‐activating peptides for membrane permeability

“…Furthermore, replacing the L isomer of Arg18 in A1 by the D isomer, to give A1(D-R18), also increased the efficacy (Green et al, 2003) (e.g. see Figure 3).…”

“…Adjacent amide backbone protons in an a-helix are separated by Bo2.8 Å and typical NOE patterns reflecting a-helical structure are shown in Figure 2. The spectrum for A2(D-R18) (Figure 2a) indicates a helix extending throughout the entire peptide from residue 1 to 20, with a helical structure comparable to that in A1(D-R18) (Green et al, 2003). Fewer NOESY cross-peaks were present in the spectrum for the active fraction 1 of A2(D-R18)-2C 10 ( Figure 2b).…”

“…The novel A2(D-R18) contained two modifications of the native A1 sequence, which increase structural stability. Ser17 was mutated to an alanine (Casarotto et al, 2000) and the L isomer of Arg18 was replaced by the D isomer (Green et al, 2003). The apparent permeabilities of A2(D-R18) and A2(D-R18)-2C 10 were measured and compared with a single tail of the same chain length, A2(D-R18)-C 10 .…”

“…A sequence of positively charged residues (El-Hayek et al, 1995;Gurrola et al, 1999;Casarotto et al, 2000Casarotto et al, , 2001, and the alignment of the residues along one surface of the molecule (Casarotto et al, 2000;Green et al, 2003), are critical for peptide A to activate RyR channels. Modified A peptides having the most stable a-helical structure are the strongest activators of the RyR and have a surface orientation of positively charged residues similar to that in two scorpion toxins, Imperatoxin A and Maurocalcine (Mosbah et al, 2000;Green et al, 2003).…”

“…Modified A peptides having the most stable a-helical structure are the strongest activators of the RyR and have a surface orientation of positively charged residues similar to that in two scorpion toxins, Imperatoxin A and Maurocalcine (Mosbah et al, 2000;Green et al, 2003). The toxins, like the peptide, are highaffinity activators of the RyR and thought to bind to a similar site on the RyR (Tripathy et al, 1998;Gurrola et al, 1999;Green et al, 2003). The A peptides are potentially useful as probes for RyR channel activity and would be more useful if they could be modified for membrane permeability without loss of function.…”