3D Mapping of the SPRY2 Domain of Ryanodine Receptor 1 by Single-Particle Cryo-EM

Alex Perálvarez-Marín,Angela F Dulhunty,Montserrat Samsó,Philip G Board,Marco G Casarotto,Hanshen Tae,Hendrik W Van Veen

doi:10.1371/journal.pone.0025813

Abstract

The type 1 skeletal muscle ryanodine receptor (RyR1) is principally responsible for Ca2+ release from the sarcoplasmic reticulum and for the subsequent muscle contraction. The RyR1 contains three SPRY domains. SPRY domains are generally known to mediate protein-protein interactions, however the location of the three SPRY domains in the 3D structure of the RyR1 is not known. Combining immunolabeling and single-particle cryo-electron microscopy we have mapped the SPRY2 domain (S1085-V1208) in the 3D structure of RyR1 using three different antibodies against the SPRY2 domain. Two obstacles for the image processing procedure; limited amount of data and signal dilution introduced by the multiple orientations of the antibody bound in the tetrameric RyR1, were overcome by modifying the 3D reconstruction scheme. This approach enabled us to ascertain that the three antibodies bind to the same region, to obtain a 3D reconstruction of RyR1 with the antibody bound, and to map SPRY2 to the periphery of the cytoplasmic domain of RyR1. We report here the first 3D localization of a SPRY2 domain in any known RyR isoform.

Highlights

RyR1 consists of 4 subunits of 565 KDa associated in a homotetramer (2.26 MDa) with fourfold symmetry
For a cryo-electron microscopy (EM) study it is important to ensure that the antibodies recognize the SPRY2 epitope in its native conformation, folded within RyR1
At least for anti-SPRY B, all this is in full agreement with the report that this antibody recognizes native RyR1 in sarcoplasmic reticulum (SR) vesicles and that it can immunoprecipitate purified RyR1 with a third of the efficacy measured for the antibody 34C [31]

Summary

Introduction

RyR1 consists of 4 subunits of 565 KDa associated in a homotetramer (2.26 MDa) with fourfold symmetry. RyR1 acts as a docking station for proteins and small molecules both in the cytosol and the sarcoplasmic reticulum (SR). Protein-protein interaction domains such as MIR, leucine zippers, EF-hands and SPRY motifs are present in RyR1, several of which are repeated along RyR1’s five thousand residue sequence [10]. The SPRY domain has been proposed as a targeting module for protein-protein interactions [11,12,13]. The SPRY motif was first identified as a repeat in the splA kinase of Dictyostelium discoideum and in the RyR sequences [14]. The generic structure of SPRY consists of a bsandwich formed by two four-stranded antiparallel b-sheets. There are three SPRY domains present in the sequence of RyR1: SPRY1 (residues 582–798), SPRY2 (residues 1085–1208), and SPRY3

Methods

Results

Conclusion