Abstract 15352: Comprehensive Analysis of Ryanodine Receptor Through Proteomic Interaction Analysis and High Resolution Crystal Structure Determination
Introduction: The ryanodine receptor is a large, homotetrameric SR membrane protein that is an essential component of calcium cycling in both skeletal and cardiac muscle. In skeletal muscle depolarization of the transverse tubular membrane triggers a conformational change in the dihydropyridine receptor (DHPR) and the ryanodine receptor type 1 (RyR1) leading to calcium release and muscle contraction. Phosphorylation is a key functional regulator of proteins and to date one phosphorylation site has been identified in RyR1 which is flanked by two RyR domains, a domain exclusively found as four repeats found as two pairs in all RyR isoforms. Here, we present the high resolution crystal structure of the two RyR domains encompassing the phosphorylation site in RyR1. We further investigate binding partners by using liquid chromatography mass spectrometry (LC-MS) and find known and novel binding partners to this region.
Methods and Results: We cloned residues 2733-2940 from RyR1 into pET28a-LIC vector containing a 6xHis tag and optimised conditions to obtain a crystal structure at 2.4Å resolution. This construct, along with other truncated RyR constructs spanning the large cytosolic regions of RyR1, were then purified using nickel beads and incubated with skeletal muscle lysate. We identified known binding partners including CaMKII and DHPR as well as potential novel binding partners such as CHERP that were then verified by co-immunoprecipitation and immunofluorescence. Functional analysis of CHERP with siRNA-mediated suppression indicated that it affects calcium release from the ER via RyR1 implying its role in the regulation of excitation-contraction coupling.
Conclusion: We have crystallised the two RyR domains that flank the only known phosphorylation site of the ryanodine receptor. This structure will help provide more detailed information into the yet unknown function of the RyR domains as well provide insight into changes that may occur in this region upon phosphorylation. Localisation and identification of known and novel binding partners throughout the large cytosolic domain of RyR1 will further help elucidate and verify key regulatory proteins and pathways involved in calcium cycling.
- © 2011 by American Heart Association, Inc.