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Report

Teaser, summary, work performed and final results

Periodic Reporting for period 1 - channelopathies (Type 1 reyanodine receptor Structure and regulation by post-translational modifications and small molecules.)

Teaser

Summary

Heart failure and other muscle-related chronic diseases, including muscular dystrophy, are often associated with high levels of circulating catecholamines and with chronic activation of adrenergic signaling pathways in many tissues. The resulting chronic adrenergic stimulation-induced PKA phosphorylation and oxidation of ryanodine receptors (RyRs)/calcium release channels at Ser2843 in the skeletal muscle RyR1 (Ser2809 in the cardiac RyR2). These protein modifications typically cause intracellular calcium leak that leads to muscle pathology including cardiac arrhythmias and skeletal muscle weakness.
To better understand the molecular mechanism of calcium release, RyR-related calcium leak, and the pathology caused by RyRs mutations in skeletal and cardiac muscles, complete models of the molecules at different states, including pathological states, are required.
Single particle cryo-EM reconstruction is a powerful tool to solve large macromolecular complexes. About 80% of the skeletal muscle RyR1 ordered structure was recently solved to ~4 Ã… resolution but did not allow unambiguous sequence assignment or side-chains orientation in most of the molecule map. Furthermore, 818 residues, at critical domains associated with disease-causing mutations were completely unresolved leaving us with a partial model that is missing valuable data. Completing the RyR1 model was and still is an important scientific goal not only to better understand cardiac and skeletal muscle diseases but also to help understand and better design drugs that can prevent fatal calcium leak, including rycals and dantrolene that specifically target RyRs.

The overall objectives were first to complete the model of the RyR1 and to understand the conformational changes that follow RyR1 PKA phosphorylation and pathological oxidation and that may lead to calcium leak and skeletal muscle damage and weakness.

Our laboratory aim to utilize, implement and develop advanced techniques to promote the understanding of mechanisms EC-coupling at the molecular and atomic level resolution and explore novel methods for understanding calcium release regulation and pathology. The study has allowed deeper understandings of how the RyR/calcium release channels are regulated, and how drugs that fix the calcium leak bind to the channels. This has important implications for developing novel therapies for heart and skeletal muscle diseases. To my knowledge, this is the first time cryo-EM was used to unambiguously draw small molecules (including drugs) binding sites. In this study we are very close to demonstrate that cryo-EM can serve as an alternative tool to crystallography for structure-based drug design, particularly targeting large protein complexes where crystallography is notoriously complicated. The strong biomedical and the translational nature of the study will make it feasible for me to rationally develop treatments targeting RyRs. As we achieved complete models of RyRs, this new information enables the determination of domain-domain interfaces and the location and structural and the functional effects of disease-causing RyRs mutations.

Work performed

In collaboration with teams at Columbia University, we now provide a near 100% complete atomic model of RyR1 based on the improved cryo-EM map obtained of the entire RyR1 molecule using focused asymmetric refinement procedure. We have also solved the structure of a RyR1-calmodulin (CaM) complex in the Ca2+/ATP-bound closed state, describing the structural basis of the RyR1-CaM interaction and providing hints as to the mechanism of inhibition. The results are at the final preparations publication of two papers that will be submitted shortly. A paper presenting the locations of all the RyR1 disease-causing mutations (image DiseaseCausingMutations.jpg) was recently published (A paper presenting nearly 100% complete model of RyR1 is in its final preparation for publication. Based on the complete model, a paper indicating the exact locations of all RyR1 disease-causing mutations (image 2) was recently published (Trends Biochem Sci. 2017, 42(7):543-555).

A method developed in my lab to label macromolecular complexes with heavy atoms clusters has proved to allow for cryo-EM data collection at close to focus conditions. We have covalently labeled surface protein cysteines with gold clusters (Au102) to increase the contrast of the labeled protein and help both identify the particles at close to zero defocus and to help align them for 2D and 3D image classifications. A map showing the strong gold cluster signal is provided (image Au102.png). This new method will be exploited in our unit to determine other small proteins structures that previously could not have been detected by Cryo-EM. Moreover, this method will also be used in the NIBN crystallography center for experimental phase determination for crystal structure determination. Once published the method and ingredients will be available to the scientific community via our website (http://lifeserv.bgu.ac.il/wp/cryoem/).

Final results

A dataset of cryo-EM single particles of RyR collected in the presence of the drug dantrolene (The primary therapy for malignant hyperthermia) has clearly identified its binding site and its effect on the channel conformation. Given the large binding-site distance from the channel pore and activation mechanism, we hypothesize that its effect is allosteric and is most likely related to the channel interaction with neighboring RyRs in the arrays forming the calcium release unit. Further experiments, involving cryo-electron tomography and CTF-corrected tomography reconstruction are being deigned to pinpoint the dantrolene effects on RyR1 arrays structures in proteoliposomes.

Using heavy atoms clusters, in particular, gold clusters that can bind surface cysteines, we were able to improve of particle peaking taken at close to zero defocus conditions. This method has a great potential to improve structure determination of macromolecules using cryo-EM and crystallography and will benefit the entire structural biology field. It may improve particle, alignment for single particle cryo-EM reconstructions and increased resolution and will allow for detection of small particles that are \'lost\' in the noise of current cryo-EM detectors. This will allow lowering the size limitation of single particle cryo-EM reconstruction. Furthermore, this method can also be beneficial for experimental phase determination of large unit cell macromolecular crystals using single or multiple wavelength anomalous difference (SAD/MAD) maps.