Molecular basis of the high-affinity activation of type 1 ryanodine receptors by imperatoxin A

Both imperatoxin A (IpTx_a), a 33-residue peptide toxin from scorpion venom, and peptide A, derived from the II-III loop of dihydropyridine receptor (DHPR), interact specifically with the skeletal ryanodine receptor (RyR1), which is a Ca²⁺-release channel in the sarcoplasmic reticulum, but with considerably different affinities. IpTx_a activates RyR1 with nanomolar affinity, whereas peptide A activates RyR1 at micromolar concentrations. To investigate the molecular basis for high-affinity activation of RyR1 by IpTx_a, we have determined the NMR solution structure of IpTx_a, and identified its functional surface by using alanine-scanning analogues. A detailed comparison of the functional surface profiles for two peptide activators revealed that IpTx_a exhibits a large functional surface area (approx. 1900 Ų, where 1 Å = 0.1 nm), based on a short double-stranded antiparallel β-sheet structure, while peptide A bears a much smaller functional surface area (approx. 800 Ų), with the five consecutive basic residues (Arg ⁶⁸¹, Lys⁶⁸², Arg⁶⁸³, Arg⁶⁸⁴ and Lys⁶⁸⁵) being clustered at the C-terminal end of the α-helix. The functional surface of IpTx_a is composed of six essential residues (Leu⁷, Lys²², Arg²³, Arg ²⁴, Arg³¹ and Arg³³) and several other important residues (His⁶, Lys⁸, Arg⁹, Lys ¹¹, Lys¹⁹, Lys²⁰, Gly²⁵, Thr ²⁶, Asn²⁷ and Lys³⁰), indicating that amino acid residues involved in RyR1 activation make up over the half of the toxin molecule with the exception of cysteine residues. Taken together, these results suggest that the site where peptide A binds to RyR1 belongs to a subset of macrosites capable of being occupied by IpTx_a, resulting in differing the affinity and the mode of activation.