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.

Striking structural variations have been reported for the receptors in different detergents and for the receptors prepared from native and sf9 cells by the same research groups, suggesting that biochemical preparations of the receptors might have had significant variations and that the heterogeneity in the samples could be a limiting factor in reaching accordant results. To resolve such discrepancies, we developed a three-layered strategy to enhance biochemical homogeneity and structural agreements. We collected two cryoEM datasets of the receptors in the absence and presence of IP3 and Ca 2þ and calculated two separate reconstructions. The two structures not only agree with each other in many aspects, but also reveal a conformational change at the top of the cytosolic domain that may lead to some reorganization of the channel pore. In order to verify the structural details and solidify the conformational changes in the pore domain, we need higher resolution structures. We are using the highend facilities with direct electron detectors to collect near-atomic resolution data in order to further improve the resolutions of our 3D reconstructions. Ryanodine receptors (RyRs) are intracellular ion channels involved in Ca 2þ release from internal stores in excitable cells. These channels are the largest channels known and are homotetramers, sizing~2,26 MDa. The 3D structure of RyR1 in it open and close states was determined previously, revealing that the ion gate opening mechanism rely on long-range conformational changes over 100 Å . The RyR gating properties are highly regulated by Ca 2þ , Mg 2þ , ATP, and FKBP12. The native conformation of RyR1 in presence of physiological concentrations of Mg 2þ and ATP is unknown. Here we determine the 3D structure of RyR1 in non-activating conditions (submicromolar Ca 2þ ) in the presence of Mg 2þ and an ATP analog, but in a flexible conformation by absence of FKPBP12. This new structure was determined using cryoEM and image processing. The resulting 3D structure is in the closed conformation when compared to 3D reconstructions of RyR1 in open and closed conditions in presence of FKBP12 determined previously. In addition, from the comparison among several 3D reconstructions, we establish new conformation-function correlations. We find that the rhomboid structures formed by domains 2-4-5-6 situated far away from the ion gate move as a whole during gating, and define a ''flexion angle'' that appears to be correlated with the degree of opening of the channel, whereby the flexion angle after adding Mg 2þ and ATP shifts by 3 degrees towards the closed state. In conclusion this research suggests that physiological concentrations of Mg 2þ and ATP shift the RyR1 conformation toward the closed conformation and also suggests that the closed conformation encompasses sub-states. 1715-Plat Crystal Structures of the Ryanodine Receptor SPRY2 DomainThe SPRY2 domain is one of three repeats of the same fold that are present within the RyR. It has been suggested as a key protein interaction site wit...

Histone lysine methyltransferases (HKMTs) are a family of epigenetic enzymes responsible for catalyzing the methylation of lysines in histones and other proteins. During the past decade, the understanding of HKMTs has significantly advanced from structural and biochemical perspectives, laying foundations for potential therapeutics against multiple diseases, in particular cancer. However, while evidence shows that HKMTs are conformationally dynamic in the context of protein methylation and inhibitor binding, limited knowledge is available about their conformational ensembles. In this work, multiple approaches were combined to explore the conformational dynamics of SETD8, which is responsible for the methylation of H4K20 and p53, and implicated in a number of cancers, such as neuroblastoma. A series of X-ray structures of SETD8 in previously unseen conformations were solved with various ligands. Molecular dynamics simulations were conducted to generate thousands of trajectories and analyzed by Markov state models to reveal the free energy landscapes, hidden conformations, and kinetic pathways between different conformations. This approach was extended to groups of HKMTs (such as NSD1/2/3, EZH1/ 2, ASH1L, SETD2) at different evolutionary distances to comparatively model the dynamics of the protein family and their functional impact. The models are analyzed to understand the free energy profiles along transitions between active and inactive conformations, the allosteric regulation of those activation profiles by protein-protein binding partners (e.g. of EZH2 in the PRC2 complex), and the role of flexible activation loops in the process. The plasticity of known binding pockets and the openings of putative allosteric pockets are analyzed, with the goal of accelerating the rational design of HKMT chemical probes through capturing unique conformations of individual enzymes.

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teeth brushing, which constantly challenge the bacterial adhesion. To survive these events, the pilus proteins contain intramolecular isopeptide bonds that confer them high mechanical stability. In the dental plaque pathogen Actinomyces oris, two strategically located isopeptide bonds in the 2 nd and the 3 rd domain of the pilus protein FimA prevent the mechanical unfolding of this protein. The only structure that can be stretched under force is the 40 residue sequence trapped between both isopeptide bonds, the isopeptide-delimited loop (IDL) motif. Our previous AFM force spectroscopy experiments unveiled the large forces (>600 pN) required to stretch FimA's IDL; however, the low force resolution of AFM prevented us from studying the IDL folding at low forces. Herein, we develop a high force magnetic tweezers force spectroscopy assay to expose FimA's IDL to forces ranging from 4 to 280 pN, allowing us to cover the IDL (un)folding dynamics with nm and sub-pN resolution. Our approach permits us to detect the IDL extension in a few seconds at forces >200 pN, and to monitor IDL folding at forces <15 pN. After IDL folding, FimA mechanical stability increases along time, suggesting a time-dependent maturation of the interfacial contacts established between FimA's 2 nd and 3 rd domains. Stretching-relaxation experiments reveal that a pilus composed of 150 FimA subunits would be able to dissipate as heat up to $4$10 5 zJ, acting as a megaDalton-scale shock-absorber. Given the ubiquitous presence of this protein motif among the pili of Gram positive bacteria, IDL pharmaceutical targeting could become a strategy to weaken the adhesion resistance of these pathogens.