bioRxiv preprint doi: https://doi.org/10.1101/2021.02.07.430153; this version posted February 10, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. Cardiac RyR N-terminal region biosensors for FRET-based high-throughput screening Jingyan Zhang1, Siobhan M. Wong King Yuen,2 Jacob A. Schwarz1, Levy M. Treinen1, Ching-Chieh Tung2, Robyn T. Rebbeck1, Kaja Berg3, Bengt Svensson1, Courtney C. Aldrich3, David D. Thomas1, Filip Van Petegem2, and Razvan L. Cornea1* 1Department of Biochemistry, Molecular Biology and Biophysics, University of Minnesota, Minneapolis, MN 55455, USA. 2Department of Biochemistry and Molecular Biology, Life Sciences Institute, University of British Columbia,V6T 1Z3 Vancouver, BC, Canada. 3Department of Medicinal Chemistry, University of Minnesota, Minneapolis, MN, 55455, USA. Corresponding author: Razvan L. Cornea Email: [email protected] Running title: FRET biosensor constructs for RyR-targeted drug discovery Keywords: Ryanodine receptor; N-terminal region; FRET; fluorescence lifetime; high-throughput screening; myopathy. bioRxiv preprint doi: https://doi.org/10.1101/2021.02.07.430153; this version posted February 10, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. Abstract failure (HF) (9-13). Physiologically, the opening and closing of RyR channels are tightly regulated The N-terminal region (NTR) of the ryanodine by small molecules, ions, and proteins(14), many receptor (RyR) calcium channels is critical to the of them binding to the enormous RyR cytoplasmic 2+ regulation of Ca release during excitation- portion, with functional effects allosterically contraction coupling. NTR hosts numerous transduced to the cytoplasmic pore through long- mutations linked to skeletal and cardiac range domain-domain interactions (15). The myopathies (RyR1 and RyR2, respectively), understanding of regulation by these domains has highlighting its potential as therapeutic target. been greatly advanced by recent high-resolution Here, we labeled the NTR of mouse RyR2 at cryo-electron microscopy (cryo-EM) RyR subdomains A, B, and C with donor and acceptor structures (15-19). The complex and rich pairs for fluorescence resonance energy transfer regulation mechanisms of RyR domains offer (FRET), obtaining two biosensors. Using opportunities to discover therapeutics that act fluorescence lifetime (FLT)-detection of either directly on RyR itself or modulate intramolecular FRET, we developed high- interacting proteins that determine RyR function. throughput screening (HTS) assays with the Aiming to discover RyR regulators for biosensors to identify small-molecule modulators therapeutic purposes, our group has previously of RyR. We screened a 1280-compound validation developed a high-throughput screening (HTS) library and identified several hits. Hits with platform based on fluorescence-labeled RyR saturable FRET dose-response profiles, and regulators FK506 binding protein 12.6 (FKBP) previously unreported effects on RyR activity, and calmodulin (CaM) (20,21). In that work, were further tested using [3H]ryanodine binding to fluorescence lifetime (FLT) detection of FRET isolated sarcoplasmic reticulum vesicles, to between the two labeled regulators was used as an measure their effects on full-length RyR opening assay to identify small-molecule modulators of in its natural membrane environment. We RyR1. Using this assay, compounds have been identified three novel inhibitors of both RyR1 and identified to decrease RyR1 Ca2+ leak in skeletal RyR2, and two RyR1-selective inhibitors at muscle SR membrane vesicles and mechanically nanomolar Ca2+. These compounds may function skinned muscle fibers (20,21), and were further as inhibitors of leaky RyRs in muscle. Two of shown to mitigate force loss in a muscular these hits activated RyR1 only at micromolar dystrophy mouse model (22). However, the Ca2+, highlighting them as potential activators of binding sites of these molecules are difficult to excitation-contraction coupling. These results identify within the full-length RyR, which limits indicate that large-scale HTS using this platform progress on understanding their mechanisms of can lead to compounds with potential for action and hinders structure-activity relationship therapeutic development. studies. Similar limitations reside with other Introduction recently reported HTS platforms for discovery using full-length of RyRs (23). An attractive The ryanodine receptor (RyR), a solution to this problem would be to develop an homotetrameric (~2.2 MDa) channel embedded in HTS platform based on constructs with the sarcoplasmic reticulum (SR) membrane, is fluorescent proteins directly attached to the RyR responsible for Ca2+ release from SR storage to (24). However, that is currently quite a cytosol, to enable excitation-contraction challenging proposition for HTS applications, coupling(1,2). Defective RyR in skeletal (RyR1) primarily due to the inadequate level of expression or cardiac (RyR2) muscle cells perturbs Ca2+ of the biosensor constructs. As a practical release behavior, which can result in acquired and alternative, we are now exploring the use of an inherited myopathies (3-9). Therefore, RyRs are essential, crystallizable RyR fragment. potential therapeutic targets for treating skeletal and cardiac myopathies, arrhythmias, and heart 2 bioRxiv preprint doi: https://doi.org/10.1101/2021.02.07.430153; this version posted February 10, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The N-terminal region (RyR residues 1-547; properties compared to the wild-type (WT)-NTR. termed NTR) is important for the functional For example, CPVT-associated mutant R420Q- regulation of RyR channels (25). The regulatory NTR lacks a Cl- ion that has been observed at a role of the NTR is partially attributed to its central location of convergence among the three structural location in the RyR tetrameric channel domains of WT-NTR. This Cl- appears to confer complex (17,26). Recent high-resolution cryo-EM RyR2 WT-NTR structural stability (35). Taken structures of the tetrameric RyR1 and RyR2, and together, the NTR is a structurally and functionally earlier docking of the NTR to lower-resolution important component of the RyR channels, and cryo-EM RyR structures, reveal that each NTR is structural perturbation of the NTR has the comprised of three domains, A, B, and C (hence potential to affect RyR channel activity. the NTR is often termed the ABC domain.). These Given the critical role of the NTR in the are located at the first tier of the RyR-channel core regulation of RyR, we have engineered NTR structure (facing the cytosol), where NTRs interact constructs for use as FRET biosensors for an HTS with each other through their respective domains platform to identify small molecules that regulate A and B, forming a tetramer that delimits a the activity of full-length RyR. We introduced vestibule leading to the RyR channel pore (Figure donor-acceptor FRET pairs at appropriately 1B) (18,27). The NTR also directly interfaces with distanced sites in different domains of the NTR the RyR bridging solenoid, and then couples with (Figure 1A), then used an FLT plate-reader to the other cytoplasmic domains to allosterically measure changes in FLT-detected FRET efficiency regulate the channel function (18). The structural as an indication of structural changes in the NTR evidence of this long-range allosteric coupling due to small-molecule binding. Initial hit between the NTR and other RyR cytoplasmic compounds obtained through FLT-FRET HTS of a domains correlates with extensive functional small library were further validated using a dose- studies (28-31), to solidify the early “domain- response format of the same FRET assay, and this zipping” hypothesis. This hypothesis postulates was followed by functional assays (ryanodine that in the normal resting state, a large N-terminal binding measurements) using full-length RyR in portion of RyR1 and RyR2 interacts with a stretch SR membrane vesicles of skeletal and cardiac of a the central portion of the RyR sequence muscles to further confirm their effects on RyR making close contact (“domain zipping”), while channels. on physiological stimulation, these critical inter- domain contacts are weakened (“domain Results unzipping”) (25), probably becoming more Engineering FRET biosensor constructs dynamic, and thus promoting channel opening based on the NTR of mouse RyR2. To build a (32). Very recently, it was also proposed that NTR FRET biosensor using isolated NTR, seven self-association is the “gatekeeper” of RyR2 solvent-exposed Cys residues were first mutated channel activity. Specifically, a stable N-terminal into Ala, to obtain a Cys-light NTR. Per construct, tetramer maintains the RyR2 channel closure, two of three residues, K30 in domain A, R383 in whereas disruption of this tetramer results in domain B, and K441 in domain C, were then channel dysfunction (33). In addition, the NTR is mutated to Cys, to provide the thiol labeling sites. one of the three major clusters of mutations Based on the crystal structure shown Figure 1, associated with skeletal and cardiac myopathies these three Cys residues are fully exposed to the such as malignant hyperthermia (MH) and solvent