Significance of Zn2+ in RyR1 for Structural Integrity and Ligand Binding: Insight from Molecular Dynamics.

Ryanodine receptor type 1 (RyR1) is a Ca2+-release channel central to skeletal muscle excitation-contraction (EC) coupling. RyR1's cryo-EM structures reveal a zinc-finger motif positioned within the cytoplasmic C-terminal domain (CTD). Yet, owing to limitations in cryo-EM resolution, RyR1 structures lack precision in detailing the metal coordination structure, prompting the need for an accurate model. In this study, we employed molecular dynamics (MD) simulations and the density functional theory (DFT) method to refine the binding characteristics of Zn2+ in the zinc-finger site of the RyR1 channel. Our findings also highlight substantial conformational changes in simulations conducted in the absence of Zn2+. Notably, we observed a loss of contact at the interface between protein domains proximal to the zinc-finger site, indicating a crucial role of Zn2+ in maintaining structural integrity and interdomain interactions within RyR1. Furthermore, this study provides valuable insights into the modulation of ATP, Ca2+, and caffeine binding, shedding light on the intricate relationship between Zn2+ coordination and the dynamic behavior of RyR1. Our integrative approach combining MD simulations and DFT calculations enhances our understanding of the molecular mechanisms governing ligand binding in RyR1.

Interplay between Mg2+ and Ca2+ at multiple sites of the ryanodine receptor.

RyR1 is an intracellular Ca2+ channel important in excitable cells such as neurons and muscle fibers. Ca2+ activates it at low concentrations and inhibits it at high concentrations. Mg2+ is the main physiological RyR1 inhibitor, an effect that is overridden upon activation. Despite the significance of Mg2+-mediated inhibition, the molecular-level mechanisms remain unclear. In this work we determined two cryo-EM structures of RyR1 with Mg2+ up to 2.8 Å resolution, identifying multiple Mg2+ binding sites. Mg2+ inhibits at the known Ca2+ activating site and we propose that the EF hand domain is an inhibitory divalent cation sensor. Both divalent cations bind to ATP within a crevice, contributing to the precise transmission of allosteric changes within the enormous channel protein. Notably, Mg2+ inhibits RyR1 by interacting with the gating helices as validated by molecular dynamics. This structural insight enhances our understanding of how Mg2+ inhibition is overcome during excitation.

Mechanisms of adaptive hypertrophic cardiac remodeling in a large animal model of premature ventricular contraction-induced cardiomyopathy.

Frequent premature ventricular contractions (PVCs) promoted eccentric cardiac hypertrophy and reduced ejection fraction (EF) in a large animal model of PVC-induced cardiomyopathy (PVC-CM), but the molecular mechanisms and markers of this hypertrophic remodeling remain unexplored. Healthy mongrel canines were implanted with pacemakers to deliver bigeminal PVCs (50% burden with 200-220 ms coupling interval). After 12 weeks, left ventricular (LV) free wall samples were studied from PVC-CM and Sham groups. In addition to reduced LV ejection fraction (LVEF), the PVC-CM group showed larger cardiac myocytes without evident ultrastructural alterations compared to the Sham group. Biochemical markers of pathological hypertrophy, such as store-operated Ca2+ entry, calcineurin/NFAT pathway, ß-myosin heavy chain, and skeletal type α-actin were unaltered in the PVC-CM group. In contrast, pro-hypertrophic and antiapoptotic pathways including ERK1/2 and AKT/mTOR were activated and/or overexpressed in the PVC-CM group, which appeared counterbalanced by an overexpression of protein phosphatase 1 and a borderline elevation of the anti-hypertrophic factor atrial natriuretic peptide. Moreover, the potent angiogenic and pro-hypertrophic factor VEGF-A and its receptor VEGFR2 were significantly elevated in the PVC-CM group. In conclusion, a molecular program is in place to keep this structural remodeling associated with frequent PVCs as an adaptive pathological hypertrophy.

Three-dimensional perspective on ryanodine receptor mutations causing skeletal and cardiac muscle-related diseases.

Mutations in RyR alter the cell's Ca2+ homeostasis and can cause serious health problems for which few effective therapies are available. Until recently, there was little structural context for the hundreds of mutations linked to muscular disorders reported for this large channel. Growing knowledge of the three-dimensional structure of RyR starts to illustrate the fine control of Ca2+ release. Current efforts directed towards understanding how disease mutations impinge in such processes will be crucial for future design of novel therapies. In this review article we discuss the up-to-date information about mutations according to their role in the 3D structure, and classified them to provide context from a structural perspective.

Alterations of sarcoplasmic reticulum-mediated Ca2+ uptake in a model of premature ventricular contraction (PVC)-induced cardiomyopathy.

Premature ventricular contractions (PVCs) are the most frequent ventricular arrhythmias in the overall population. PVCs are known to acutely enhance contractility by the post-extrasystolic potentiation phenomenon, but over time persistent PVCs promote PVC-induced cardiomyopathy (PVC-CM), characterized by a reduction of the left ventricular (LV) ejection fraction. Ca2+ cycling in myocytes commands muscle contraction and in this process, SERCA2 leads the Ca2+ reuptake into the sarcoplasmic reticulum (SR) shaping cytosolic Ca2+ signal decay and muscle relaxation. Altered Ca2+ reuptake can contribute to the contractile dysfunction observed in PVC-CM. To better understand Ca2+ handling using our PVC-CM model (canines with 50% PVC burden for 12 weeks), SR-Ca2+ reuptake was investigated by measuring Ca2+ dynamics and analyzing protein expression. Kinetic analysis of Ca2+ reuptake in electrically paced myocytes showed a ~ 21 ms delay in PVC-CM compared to Sham in intact isolated myocytes, along with a ~ 13% reduction in SERCA2 activity assessed in permeabilized myocytes. Although these trends were not statistically significant between groups using hierarchical statistics, relaxation of myocytes following contraction was significantly slower in PVC-CM vs Sham myocytes. Western blot analyses indicate a 22% reduction in SERCA2 expression, a 23% increase in phospholamban (PLN) expression, and a 50% reduction in PLN phosphorylation in PVC-CM samples vs Sham. Computational analysis simulating a 20% decrease in SR-Ca2+ reuptake resulted in a ~ 22 ms delay in Ca2+ signal decay, consistent with the experimental result described above. In conclusion, SERCA2 and PLB alterations described above have a modest contribution to functional adaptations observed in PVC-CM.

Cryo-EM Structure of the Type IV Pilus Extension ATPase from Enteropathogenic Escherichia coli.

Type 4 pili (T4P) are retractable surface appendages found on numerous bacteria and archaea that play essential roles in various microbial functions, including host colonization by pathogens. An ATPase is required for T4P extension, but the mechanism by which chemical energy is transduced to mechanical energy for pilus extension has not been elucidated. Here, we report the cryo-electron microscopy (cryo-EM) structure of the BfpD ATPase from enteropathogenic Escherichia coli (EPEC) in the presence of either ADP or a mixture of ADP and AMP-PNP. Both structures, solved at 3 Å resolution, reveal the typical toroid shape of AAA+ ATPases and unambiguous 6-fold symmetry. This 6-fold symmetry contrasts with the 2-fold symmetry previously reported for other T4P extension ATPase structures, all of which were from thermophiles and solved by crystallography. In the presence of the nucleotide mixture, BfpD bound exclusively AMP-PNP, and this binding resulted in a modest outward expansion in comparison to the structure in the presence of ADP, suggesting a concerted model for hydrolysis. De novo molecular models reveal a partially open configuration of all subunits where the nucleotide binding site may not be optimally positioned for catalysis. ATPase functional studies reveal modest activity similar to that of other extension ATPases, while calculations indicate that this activity is insufficient to power pilus extension. Our results reveal that, despite similarities in primary sequence and tertiary structure, T4P extension ATPases exhibit divergent quaternary configurations. Our data raise new possibilities regarding the mechanism by which T4P extension ATPases power pilus formation. IMPORTANCE Type 4 pili are hairlike surface appendages on many bacteria and archaea that can be extended and retracted with tremendous force. They play a critical role in disease caused by several deadly human pathogens. Pilus extension is made possible by an enzyme that converts chemical energy to mechanical energy. Here, we describe the three-dimensional structure of such an enzyme from a human pathogen in unprecedented detail, which reveals a mechanism of action that has not been seen previously among enzymes that power type 4 pilus extension.

Molecular mechanism of the severe MH/CCD mutation Y522S in skeletal ryanodine receptor (RyR1) by cryo-EM.

Ryanodine receptors (RyRs) are main regulators of intracellular Ca2+ release and muscle contraction. The Y522S mutation of RyR1 causes central core disease, a weakening myopathy, and malignant hyperthermia, a sudden and potentially fatal response to anesthetics or heat. Y522 is in the core of the N-terminal subdomain C of RyR1 and the mechanism of how this mutation orchestrates malfunction is unpredictable for this 2-MDa ion channel, which has four identical subunits composed of 15 distinct cytoplasmic domains each. We expressed and purified the RyR1 rabbit homolog, Y523S, from HEK293 cells and reconstituted it in nanodiscs under closed and open states. The high-resolution cryogenic electron microscopic (cryo-EM) three-dimensional (3D) structures show that the phenyl ring of Tyr functions in a manner analogous to a "spacer" within an α-helical bundle. Mutation to the much smaller Ser alters the hydrophobic network within the bundle, triggering rearrangement of its α-helices with repercussions in the orientation of most cytoplasmic domains. Examining the mutation-induced readjustments exposed a series of connected α-helices acting as an ∼100 Å-long lever: One end protrudes toward the dihydropyridine receptor, its molecular activator (akin to an antenna), while the other end reaches the Ca2+ activation site. The Y523S mutation elicits channel preactivation in the absence of any activator and full opening at 1.5 µM free Ca2+, increasing by ∼20-fold the potency of Ca2+ to activate the channel compared with RyR1 wild type (WT). This study identified a preactivated pathological state of RyR1 and a long-range lever that may work as a molecular switch to open the channel.

Ca2+ inactivation of the mammalian ryanodine receptor type 1 in a lipidic environment revealed by cryo-EM.

Activation of the intracellular Ca2+ channel ryanodine receptor (RyR) triggers a cytosolic Ca2+ surge, while elevated cytosolic Ca2+ inhibits the channel in a negative feedback mechanism. Cryogenic electron microscopy of rabbit RyR1 embedded in nanodiscs under partially inactivating Ca2+ conditions revealed an open and a closed-inactivated conformation. Ca2+ binding to the high-affinity site engages the central and C-terminal domains into a block, which pries the S6 four-helix bundle open. Further rotation of this block pushes S6 toward the central axis, closing (inactivating) the channel. Main characteristics of the Ca2+-inactivated conformation are downward conformation of the cytoplasmic assembly and tightly knit subunit interface contributed by a fully occupied Ca2+ activation site, two inter-subunit resolved lipids, and two salt bridges between the EF hand domain and the S2-S3 loop validated by disease-causing mutations. The structural insight illustrates the prior Ca2+ activation prerequisite for Ca2+ inactivation and provides for a seamless transition from inactivated to closed conformations.

Purification of Recombinant Wild Type and Mutant Ryanodine Receptors Expressed in HEK293 Cells.

High quantities of purified ryanodine receptor (RyR), a large (2.26 MDa) intracellular homotetrameric membrane protein, can be obtained from heterologous expression in HEK293 cells and used for structure determination by cryo-EM. The advantage of using recombinant protein is that the variability due to post-translational modifications can be minimized, to which the high resolution of up to 2.4 Å achieved for RyR2 can be attributed ( Iyer et al., 2020 ). In addition, recombinant protein expression enables the study of mutations that are deleterious when expressed homozygously in animals. Protein purification was achieved using two strategies, sucrose density gradient and affinity chromatography, which have previously been used for purification of RyR from tissue. The sucrose gradient method was developed from ( Lee et al., 1994 ) and later adapted for cryo-EM ( Samsó et al., 2005 ). The affinity chromatography method takes advantage of the high affinity of RyR for its ligand FKBP12/12.6, by using a construct between FKBP and streptavidin binding protein (SBP) ( Cabra et al., 2016 ). While the sucrose gradient method can yield a higher protein concentration (≥ 2 mg/ml), the affinity purification method is faster. Both methods are suitable and applicable to the purification of recombinant proteins and were successfully used in the first 3D near-atomic reconstructions of RyRs purified from cells expressing disease mutants ( Iyer et al., 2020 ). This purification protocol is also suitable for functional studies, such as single-channel analysis, that require pure RyR protein.

Structural mechanism of two gain-of-function cardiac and skeletal RyR mutations at an equivalent site by cryo-EM.

Mutations in ryanodine receptors (RyRs), intracellular Ca2+ channels, are associated with deadly disorders. Despite abundant functional studies, the molecular mechanism of RyR malfunction remains elusive. We studied two single-point mutations at an equivalent site in the skeletal (RyR1 R164C) and cardiac (RyR2 R176Q) isoforms using ryanodine binding, Ca2+ imaging, and cryo-electron microscopy (cryo-EM) of the full-length protein. Loss of the positive charge had greater effect on the skeletal isoform, mediated via distortion of a salt bridge network, a molecular latch inducing rotation of a cytoplasmic domain, and partial progression to open-state traits of the large cytoplasmic assembly accompanied by alteration of the Ca2+ binding site, which concur with the major "hyperactive" feature of the mutated channel. Our cryo-EM studies demonstrated the allosteric effect of a mutation situated ~85 Å away from the pore and identified an isoform-specific structural effect.

The FKBP12 subunit modifies the long-range allosterism of the ryanodine receptor.

Ryanodine receptors (RyRs) are large conductance intracellular channels controlling intracellular calcium homeostasis in myocytes, neurons, and other cell types. Loss of RyR's constitutive cytoplasmic partner FKBP results in channel sensitization, dominant subconductance states, and increased cytoplasmic Ca2+. FKBP12 binds to RyR1's cytoplasmic assembly 130 Šaway from the ion gate at four equivalent sites in the RyR1 tetramer. To understand how FKBP12 binding alters RyR1's channel properties, we studied the 3D structure of RyR1 alone in the closed conformation in the context of the open and closed conformations of FKBP12-bound RyR1. We analyzed the metrics of conformational changes of existing structures, the structure of the ion gate, and carried out multivariate statistical analysis of thousands of individual cryoEM RyR1 particles. We find that under closed state conditions, in the presence of FKBP12, the cytoplasmic domain of RyR1 adopts an upward conformation, whereas absence of FKBP12 results in a relaxed conformation, while the ion gate remains closed. The relaxed conformation is intermediate between the RyR1-FKBP12 complex closed (upward) and open (downward) conformations. The closed-relaxed conformation of RyR1 appears to be consistent with a lower energy barrier separating the closed and open states of RyR1-FKBP12, and suggests that FKBP12 plays an important role by restricting conformations within RyR1's conformational landscape.

Hyaluronic Acid Grafted Nanoparticles of a Platinum(II)-Silicon(IV) Phthalocyanine Conjugate for Tumor and Mitochondria-Targeted Photodynamic Therapy in Red Light.

Herein, we report novel hyaluronic acid formulated nanoparticles containing a platinum(II) conjugated silicon(IV) phthalocyanine (SiPc-Pt-HA) for tumor targeted red light photodynamic therapy and chemotherapy. The SiPc-Pt-HA conjugate showed specific uptake, photo-enhanced cytotoxicity (~1500 fold) and mitochondrial accumulation in breast cancer over normal cells.

A cryo-EM-based model of phosphorylation- and FKBP12.6-mediated allosterism of the cardiac ryanodine receptor.

Type 2 ryanodine receptors (RyR2s) are calcium channels that play a vital role in triggering cardiac muscle contraction by releasing calcium from the sarcoplasmic reticulum into the cytoplasm. Several cardiomyopathies are associated with the abnormal functioning of RyR2. We determined the three-dimensional structure of rabbit RyR2 in complex with the regulatory protein FKBP12.6 in the closed state at 11.8 Å resolution using cryo-electron microscopy and built an atomic model of RyR2. The heterogeneity in the data set revealed two RyR2 conformations that we proposed to be related to the extent of phosphorylation of the P2 domain. Because the more flexible conformation may correspond to RyR2 with a phosphorylated P2 domain, we suggest that phosphorylation may set RyR2 in a conformation that needs less energy to transition to the open state. Comparison of RyR2 from cardiac muscle and RyR1 from skeletal muscle showed substantial structural differences between the two, especially in the helical domain 2 (HD2) structure forming the Clamp domain, which participates in quaternary interactions with the dihydropyridine receptor and neighboring RyRs in RyR1 but not in RyR2. Rigidity of the HD2 domain of RyR2 was enhanced by binding of FKBP12.6, a ligand that stabilizes RyR2 in the closed state. These results help to decipher the molecular basis of the different mechanisms of activation and oligomerization of the RyR isoforms and could be extended to RyR complexes in other tissues.

A guide to the 3D structure of the ryanodine receptor type 1 by cryoEM.

Signal transduction by the ryanodine receptor (RyR) is essential in many excitable cells including all striated contractile cells and some types of neurons. While its transmembrane domain is a classic tetrameric, six-transmembrane cation channel, the cytoplasmic domain is uniquely large and complex, hosting a multiplicity of specialized domains. The overall outline and substructure readily recognizable by electron microscopy make RyR a geometrically well-behaved specimen. Hence, for the last two decades, the 3D structural study of the RyR has tracked closely the technological advances in electron microscopy, cryo-electron microscopy (cryoEM), and computerized 3D reconstruction. This review summarizes the progress in the structural determination of RyR by cryoEM and, bearing in mind the leap in resolution provided by the recent implementation of direct electron detection, analyzes the first near-atomic structures of RyR. These reveal a complex orchestration of domains controlling the channel's function, and help to understand how this could break down as a consequence of disease-causing mutations.

Ultrastructural Analysis of Self-Associated RyR2s.

In heart, type-2 ryanodine receptor (RyR2) forms discrete supramolecular clusters in the sarcoplasmic reticulum known as calcium release units (CRUs), which are responsible for most of the Ca(2+) released for muscle contraction. To learn about the substructure of the CRU, we sought to determine whether RyR2s have the ability to self-associate in the absence of other factors and if so, whether they do it in a specific manner. Purified RyR2 was negatively stained and imaged on the transmission electron microscope, and RyR2 particles closely associated were further analyzed using bias-free multivariate statistical analysis and classification. The resulting two-dimensional averages show that RyR2s can interact in two rigid, reproducible configurations: "adjoining", with two RyR2s alongside each other, and "oblique", with two partially overlapped RyR2s forming an angle of 12°. The two configurations are nearly identical under two extreme physiological Ca(2+) concentrations. Pseudo-atomic models for these two interactions indicate that the adjoining interaction involves contacts between the P1, SPRY1 and the helical domains. The oblique interaction is mediated by extensive contacts between the SPRY1 domains (domains 9) and P1 domains (domains 10) of both RyR2s and not through domain 6 as previously thought; in addition its asymmetric interface imposes steric constrains that inhibit the growth of RyR2 as a checkerboard, which is the configuration usually assumed, and generates new configurations, i.e., "branched" and "interlocked". This first, to our knowledge, structural detailed analysis of the inter-RyR2 interactions helps to understand important morphological and functional aspects of the CRU in the context of cardiac EC coupling.

Do's and don'ts of cryo-electron microscopy: a primer on sample preparation and high quality data collection for macromolecular 3D reconstruction.

Cryo-electron microscopy (cryoEM) entails flash-freezing a thin layer of sample on a support, and then visualizing the sample in its frozen hydrated state by transmission electron microscopy (TEM). This can be achieved with very low quantity of protein and in the buffer of choice, without the use of any stain, which is very useful to determine structure-function correlations of macromolecules. When combined with single-particle image processing, the technique has found widespread usefulness for 3D structural determination of purified macromolecules. The protocol presented here explains how to perform cryoEM and examines the causes of most commonly encountered problems for rational troubleshooting; following all these steps should lead to acquisition of high quality cryoEM images. The technique requires access to the electron microscope instrument and to a vitrification device. Knowledge of the 3D reconstruction concepts and software is also needed for computerized image processing. Importantly, high quality results depend on finding the right purification conditions leading to a uniform population of structurally intact macromolecules. The ability of cryoEM to visualize macromolecules combined with the versatility of single particle image processing has proven very successful for structural determination of large proteins and macromolecular machines in their near-native state, identification of their multiple components by 3D difference mapping, and creation of pseudo-atomic structures by docking of x-ray structures. The relentless development of cryoEM instrumentation and image processing techniques for the last 30 years has resulted in the possibility to generate de novo 3D reconstructions at atomic resolution level.

3D Structure of the Dihydropyridine Receptor of Skeletal Muscle.

Excitation contraction coupling, the rapid and massive Ca(2+) release under control of an action potential that triggers muscle contraction, takes places at specialized regions of the cell called triad junctions. There, a highly ordered supramolecular complex between the dihydropyridine receptor (DHPR) and the ryanodine receptor (RyR1) mediates the quasi-instantaneous conversion from T-tubule depolarization into Ca(2+) release from the sarcoplasmic reticulum (SR). The DHPR has several key modules required for EC coupling: the voltage sensors and II-III loop in the alpha1s subunit, and the beta subunit. To gain insight into their molecular organization, this review examines the most updated 3D structure of the DHPR as obtained by transmission electron microscopy and image reconstruction. Although structure determination of a heteromeric membrane protein such as the DHPR is challenging, novel technical advances in protein expression and 3D labeling facilitated this task. The 3D structure of the DHPR complex consists of a main body with five irregular corners around its perimeter encompassing the transmembrane alpha 1s subunit besides the intracellular beta subunit, an extended extracellular alpha 2 subunit, and a bulky intracellular II-III loop. The structural definition attained at 19 Å resolution enabled docking of the atomic coordinates of structural homologs of the alpha1s and beta subunits. These structural features, together with their relative location with respect to the RyR1, are discussed in the context of the functional data.

The ArrayGrid: a methodology for applying multiple samples to a single TEM specimen grid.

High throughput transmission electron microscopy (TEM) is limited by the time that it takes to prepare each specimen and insert it on the microscope. It is further impeded by the deteriorating vacuum of the microscope upon frequent specimen cycling. Nevertheless, in most cases only a small fraction of the specimen is examined and sufficient to provide hundreds of images. Here we demonstrate that microarray technology can be used to accurately position picoliter quantities of different samples in a single TEM grid, with negligible cross-contamination. Key features are a contact-mode deposition on a robust formvar-carbon support. The TEM grid containing a microarray of different samples, the ArrayGrid, can also be negatively stained. The ArrayGrid increases the efficiency of TEM grid preparation and examination by at least by one order of magnitude, and is very suitable for screening and data collection especially in experiments that generate a multiplicity of samples.

Structural determinants of skeletal muscle ryanodine receptor gating.

Ryanodine receptor type 1 (RyR1) releases Ca(2+) from intracellular stores upon nerve impulse to trigger skeletal muscle contraction. Effector binding at the cytoplasmic domain tightly controls gating of the pore domain of RyR1 to release Ca(2+). However, the molecular mechanism that links effector binding to channel gating is unknown due to lack of structural data. Here, we used a combination of computational and electrophysiological methods and cryo-EM densities to generate structural models of the open and closed states of RyR1. Using our structural models, we identified an interface between the pore-lining helix (Tyr-4912-Glu-4948) and a linker helix (Val-4830-Val-4841) that lies parallel to the cytoplasmic membrane leaflet. To test the hypothesis that this interface controls RyR1 gating, we designed mutations in the linker helix to stabilize either the open (V4830W and T4840W) or closed (H4832W and G4834W) state and validated them using single channel experiments. To further confirm this interface, we designed mutations in the pore-lining helix to stabilize the closed state (Q4947N, Q4947T, and Q4947S), which we also validated using single channel experiments. The channel conductance and selectivity of the mutations that we designed in the linker and pore-lining helices were indistinguishable from those of WT RyR1, demonstrating our ability to modulate RyR1 gating without affecting ion permeation. Our integrated computational and experimental approach significantly advances the understanding of the structure and function of an unusually large ion channel.