Structural Studies of the Ryanodine Receptor and Its Binding Protein, Calstabin
- Structural Studies of the Ryanodine Receptor and Its Binding Protein, Calstabin
- Duvshani, Amit
- Thesis Advisor(s):
- Marks, Andrew
- Ph.D., Columbia University
- Physiology and Cellular Biophysics
- Persistent URL:
- The ryanodine receptor (RyR) is a heterotetrameric Ca2+ release channel located on the sarcoplasmic reticulum (SR) membrane of different cell types. RyR type 1 (RyR1) is the dominant isoform in skeletal muscle and RyR type 2 (RyR2) is abundant in the heart. The RyR N-terminus is a large cytoplasmic domain that binds many channel modulators, including the immunophilin calstabin.
Calstabins (FKBPs) -- which are cis-trans peptydil-prolyl isomerases -- modify and bind to RyRs. Calstabin1 (FKBP12) is associated with RyR1 and calstabin2 (FKBP12.6) binds to RyR2. The binding site for calstabins on RyRs has been studied and includes a proline. The proline is preceded by a valine or an isoleucine in both RyR isoforms. Calstabins bind to the immunosuppressive drugs rapamycin and FK506; this binding suppresses the isomerase activity of these drugs. It has been proposed that this inhibition is caused by the ability of the immunosuppressive compounds to mimic the transition state of ligand isomerization.
RyR undergoes several types of post-translational modifications. One of these modifications, phosphorylation by protein kinase A (PKA) at Ser2808, causes a decrease in affinity of calstabin to the channel. The dissociation of calstabin from the channel increases channel openings and promotes sub-conductance states. This phenomenon causes Ca2+ `leak' from the SR into the cytoplasm and depletes the Ca2+ stores of the cell. The aberrant release of Ca2+ can promote different disease states. For example, SR Ca2+ leak in cardiac cells can promote heart failure (HF) and fatal ventricular arrhythmias.
The Marks lab demonstrated that a calstabin2 mutant -- in which Asp37 was mutated into valine -- retained the ability to bind to PKA-phosphorylated channels. Single channel measurements have shown that binding of the calstabin2-D37V restored the calstabin2-bound channel properties.
In the present study we aimed to structurally understand the differences in binding between wt-calstabin2 and D37V-calstabin2. To this end, we cloned, expressed and purified the D37V-calstabin2 with an MBP fusion protein. The fusion protein was crystallized in the presence of rapamycin and the structure was solved using molecular replacement techniques. The main difference between the mutant and wt calstabin2 was that a hydrogen bond between D37 and rapamycin was replaced with a van der Waals interaction.
We also docked the mutant calstabin2-D37V into our cryo-EM structure of RyR1. We were able to clearly see that the amino acids D (or V) interacted with a helix projecting from the RyR structure, which we believe to contain the proline previously identified by the Marks group. Calstabin2 interacted with the receptor via three distinct domains; this interaction has implications for coupled gating, phosphorylation and disease-associated mutations.
The binding affinity of the wt and mutant calstabins was measured using radiolabeled versions of wt and D37V proteins. We found that the affinity of wt calstabin2 to PKA-phosphorylated RyR2 decreased threefold compared to non-phosphorylated RyR. The D37V mutant, however, was able to bind to both phosphorylated and non-phosphorylated RyR2 with the same affinity.
This study also included efforts to crystallize different RyR fragments. We attempted to crystallize RyR1 and RyR2 domains that are involved in RyR regulation by small modulators or domains that are important to its activity. Despite not being able to crystallize these fragments, we present our results here and suggest they could serve us in the future for a variety of biochemical and biophysical studies.
- Item views
text | xml
- Suggested Citation:
- Amit Duvshani, 2014, Structural Studies of the Ryanodine Receptor and Its Binding Protein, Calstabin, Columbia University Academic Commons, https://doi.org/10.7916/D8VD6WM1.