Structural studies of selected human ryanodine receptor 2 domains

Vladena Bauerová¹, Eva Hostinová¹, Juraj Gašperík¹, Lubomír Borko¹, Matúš Hajduk¹, Alexandra Zahradníková^2,1 and Jozef Ševčík¹

¹ Institute of Molecular Biology SAS, Dúbravská cesta 21, 845 51 Bratislava

² Institute of Molecular Physiology and Genetics SAS, Vlárska 5, 833 34 Bratislava

Human ryanodine receptor 2 (RyR2) is one of the three isoforms of the ryanodine receptor, the ion channel that mediates Ca²⁺ release from intracellular calcium stores. RyR2 is responsible for releasing Ca²⁺ ions necessary for contraction in cardiac myocytes. It is a large homotetramer, composed of four ~5000 amino acid residues. Because the whole molecule is very large, we identified, cloned, expressed and purified individual domains of RyR2 and performed first crystallization experiments.

The first domain prediction, obtained by program Pfam, suggested the existence of 14 domains in the RyR monomer. The prediction was further adjusted by comparison with RyR2 secondary structure prediction and with the 3D structure of the IP3-binding domain of the inositol-3-phosphate receptor (PDB ID 1N4K). The cDNA sequence involving residues 1 - 759 (Tunwell et al., Biochem J. 318:477-487, 1996) was obtained from Prof. F.A. Lai (University of Cardiff). A fragment from the central part of the RyR was synthesized by GeneScript, USA. Three N-terminal domains and two central domains were cloned and expressed with His-Tag fusion at C-terminus. The N-terminal domains were also expressed with Nus or Trx fusion partners to increase solubility and obtain correct folding of expressed proteins. All constructs were expressed in E. coli, BL21 (DE3) at optimized temperature, IPTG concentration and expression time. His-Tag purification was used as the main purification step. The monomeric state of each purified fragment was assessed by gel filtration. The tested N-terminal domains differed markedly in their expression level, solubility and monomeric state. Crystallization experiments with one of the fragments, performed with the crystallization kit Structure Screen 1 (Molecular Dimensions), yielded crystals with dimensions ~0.2 x 0.1 x 0.05 in 1.5 M Lithium sulfate, 2% PEG 8000. Crystallization of other fragments and testing their diffracting power is under way.

This work was supported by the grant APVV-0139-06 from the Slovak Research and Development Agency.