Three-dimensional reconstruction of the recombinant type 3 ryanodine receptor and localization of its amino terminus

• DOI: 10.1073/pnas.111382798
• Publication Date: 2002-07-26T14:44:19Z
• Authors: Zheng Liu; Jing Zhang; Manjuli R. Sharma; Pin Li; S. R. Wayne Chen; Terence Wagenknecht
• Keywords: 0303 health sciences; 03 medical and health sciences; Protein Conformation; Recombinant Fusion Proteins; Cryoelectron Microscopy; Humans; Ryanodine Receptor Calcium Release Channel; Cell Line; Glutathione Transferase

Recombinant type 3 ryanodine receptor (RyR3) has been purified in quantities sufficient for structural characterization by cryoelectron microscopy and three-dimensional (3D) reconstruction. Two cDNAs were prepared and expressed in HEK293 cells, one encoding the wild-type RyR3 and the other encoding RyR3 containing glutathione S -transferase (GST) fused to its amino terminus (GST-RyR3). RyR3 was purified from detergent-solubilized transfected cells by affinity chromatography using 12.6-kDa FK506-binding protein in the form of a GST fusion as the affinity ligand. Purification of GST-RyR3 was achieved by affinity chromatography by using glutathione-Sepharose. Purified recombinant RyR3 and GST-RyR3 proteins exhibited high-affinity [ 3 H]ryanodine binding that was sensitive to activation by Ca 2+ and caffeine and to inhibition by Mg 2+ . 3D reconstructions of both recombinant RyR3 and GST-RyR3 appeared very similar to that of the native RyR3 purified from bovine diaphragm. Comparison of the 3D reconstructions of RyR3 and GST-RyR3 revealed that the GST domains and, hence, the amino termini of the RyR3 subunits are located in the “clamp” structures that form the corners of the square-shaped cytoplasmic region of homotetrameric RyR3. This study describes the 3D reconstruction of a recombinant ryanodine receptor and it demonstrates the potential of this technology for characterizing functional and structural perturbations introduced by site-directed mutagenesis.