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Molecular recognition of the disordered dihydropyridine receptor II-III loop by a conserved domain in the type 1 ryanodine receptor

Y. Cui, H-S. Tae, N.C. Norris, Y. Karunasekara, P. Pouliquin, P.G. Board, A.F. Dulhunty and M.G. Casarotto, John Curtin School of Medical Research, Australian National University, ACT 0200, Australia.

Conformational coupling between the dihydropyridine receptor (DHPR) and the skeletal ryanodine receptor (RyR1) is essential for excitation-contraction (EC) coupling in skeletal muscle. The II-III loop of the DHPR α1s subunit is central to this coupling process, but neither its structure nor its mode of binding to RyR1 are known. We have investigated the NMR-derived structure of the α1s II-III loop and its binding to a region of the skeletal RyR1. We find that the II-III loop is highly flexible, with a strong N-terminal helix followed by several nascent helical/turn elements and unstructured segments, but it possesses no stable tertiary fold. The II-III loop thus belongs to a burgeoning class of functionally important, intrinsically unstructured, proteins. We have mapped the area of II-III loop interaction with RyR1 as a SPRY domain (1085-1208) and have identified which regions of II-III loop that are directly involved in binding by NMR methods. The principle site of interaction is through the N-terminal helix (A region, 671-690) but the B region (691-723) is also found to participate in binding. Confirmation that the A region of the II-III loop is indeed involved in SPRY2 binding is demonstrated through a series of mutations that clearly implicate a stretch of basic residues (R681-K685) as an important structural determinant. Evidence was found suggesting that there are weaker interactions between the C region of the loop and SPRY2. We propose that the flexible nature of the II-III loop is required for segments of the loop to associate and disassociate with RyR1 when the surface membrane potential changes, so that the loop may act as a conformational switch in EC coupling.