Publications by authors named "Filip van Petegem"

90 Publications

Distinct protein architectures mediate species-specific beta-glucan binding and metabolism in the human gut microbiota.

J Biol Chem 2021 Feb 12:100415. Epub 2021 Feb 12.

Michael Smith Laboratories, University of British Columbia, 2185 East Mall, Vancouver, BC, V6T 1Z4, Canada; Department of Biochemistry and Molecular Biology, the Life Sciences Institute, University of British Columbia, 2350 Health Sciences Mall, Vancouver, BC, V6T 1Z3, Canada; Department of Chemistry, University of British Columbia, 2036 Main Mall, Vancouver, BC, V6T 1Z1, Canada. Electronic address:

Complex glycans that evade our digestive system are major nutrients that feed the human gut microbiota (HGM). The prevalence of Bacteroidetes in the HGM of populations worldwide is engendered by the evolution of Polysaccharide Utilization Locus (PUL), which encode concerted protein systems to utilize the myriad complex glycans in our diets. Despite their crucial roles in glycan recognition and transport, cell-surface glycan-binding proteins (SGBPs) remained understudied cogs in the PUL machinery. Here, we report the structural and biochemical characterization of a suite of SGBP-A and SGBP-B structures from three syntenic β(1,3)-glucan utilization loci (1,3GULs) from Bacteroides thetaiotaomicron (Bt), B. uniformis (Bu), and B. fluxus (Bf), which have varying specificities for distinct β-glucans. Ligand complexes provide definitive insight into β(1,3)-glucan selectivity in the HGM, including structural features enabling dual β(1,3)-glucan/mixed-linkage β(1,3)/β(1,4)-glucan binding capability in some orthologs. The tertiary structural conservation of SusD-like SGBPs-A is juxtaposed with the diverse architectures and binding modes of the SGBPs-B. Specifically, the structures of the tri-modular BtSGBP-B and BuSGBP-B revealed a tandem repeat of Carbohydrate-Binding Module-Like domains connected by long linkers. In contrast, BfSGBP-B comprises a bi-modular architecture with a distinct β-barrel domain at the C-terminus that bears a shallow binding canyon. The molecular insights obtained here contribute to our fundamental understanding of HGM function, which in turn may inform tailored microbial intervention therapies.
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http://dx.doi.org/10.1016/j.jbc.2021.100415DOI Listing
February 2021

Binding of calcium and magnesium to human cardiac Troponin C.

J Biol Chem 2021 Feb 3:100350. Epub 2021 Feb 3.

Molecular Cardiac Physiology Group, Simon Fraser University, Burnaby, BC, V5A 1S6, Canada; Department of Molecular Biology and Biochemistry, Simon Fraser University, Burnaby, BC, V5A 1S6, Canada; BC Children's Hospital Research Institute, Vancouver, BC, V5Z 4H4, Canada. Electronic address:

Cardiac muscle thin filaments are composed of actin, tropomyosin, and troponin that change conformation in response to Ca binding, triggering muscle contraction. Human cardiac troponin C (cTnC) is the Ca-sensing component of the thin filament. It contains structural sites (III/IV) that bind both Ca and Mg, and a regulatory site (II) that has been thought to bind only Ca. Binding of Ca at this site initiates a series of conformational changes that culminate in force production. However, the mechanisms that underpin the regulation of binding at site II remain unclear. Here, we have quantified the interaction between site II and Ca/Mg through Isothermal Titration Calorimetry and Thermodynamic Integration simulations. Direct and competitive binding titration with wild type N-terminal cTnC (N-cTnC) and full-length cTnC indicate that physiologically relevant concentrations of both Ca/ Mg interacted with the same locus. Moreover, the D67A/D73A N-cTnC construct in which two coordinating residues within site II were removed was found to have significantly reduced affinity for both cations. In addition, 1 mM Mg caused a 1.4-fold lower affinity for Ca. These experiments strongly suggest that cytosolic free Mg occupies a significant population of the available site II. Interaction of Mg with site II of cTnC likely has important functional consequences for the heart both at baseline as well as in diseased states which decrease or increase the availability of Mg such as secondary hyperparathyroidism or ischemia, respectively.
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http://dx.doi.org/10.1016/j.jbc.2021.100350DOI Listing
February 2021

Pathological conformations of disease mutant Ryanodine Receptors revealed by cryo-EM.

Nat Commun 2021 02 5;12(1):807. Epub 2021 Feb 5.

University of British Columbia, Department of Biochemistry and Molecular Biology, Life Sciences Centre, Vancouver, BC, Canada.

Ryanodine Receptors (RyRs) are massive channels that release Ca from the endoplasmic and sarcoplasmic reticulum. Hundreds of mutations are linked to malignant hyperthermia (MH), myopathies, and arrhythmias. Here, we explore the first MH mutation identified in humans by providing cryo-EM snapshots of the pig homolog, R615C, showing that it affects an interface between three solenoid regions. We also show the impact of apo-calmodulin (apoCaM) and how it can induce opening by bending of the bridging solenoid, mediated by its N-terminal lobe. For R615C RyR1, apoCaM binding abolishes a pathological 'intermediate' conformation, distributing the population to a mixture of open and closed channels, both different from the structure without apoCaM. Comparisons show that the mutation primarily affects the closed state, inducing partial movements linked to channel activation. This shows that disease mutations can cause distinct pathological conformations of the RyR and facilitate channel opening by disrupting interactions between different solenoid regions.
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http://dx.doi.org/10.1038/s41467-021-21141-3DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7864917PMC
February 2021

Sorcin is an early marker of neurodegeneration, Ca dysregulation and endoplasmic reticulum stress associated to neurodegenerative diseases.

Cell Death Dis 2020 10 15;11(10):861. Epub 2020 Oct 15.

Institute of Molecular Biology and Pathology, Italian National Research Council, IBPM-CNR, Rome, Italy.

Dysregulation of calcium signaling is emerging as a key feature in the pathogenesis of neurodegenerative diseases such as Alzheimer's disease (AD), Parkinson's disease (PD), and Huntington's disease (HD), and targeting this process may be therapeutically beneficial. Under this perspective, it is important to study proteins that regulate calcium homeostasis in the cell. Sorcin is one of the most expressed calcium-binding proteins in the human brain; its overexpression increases endoplasmic reticulum (ER) calcium concentration and decreases ER stress in the heart and in other cellular types. Sorcin has been hypothesized to be involved in neurodegenerative diseases, since it may counteract the increased cytosolic calcium levels associated with neurodegeneration. In the present work, we show that Sorcin expression levels are strongly increased in cellular, animal, and human models of AD, PD, and HD, vs. normal cells. Sorcin partially colocalizes with RyRs in neurons and microglia cells; functional experiments with microsomes containing high amounts of RyR2 and RyR3, respectively, show that Sorcin is able to regulate these ER calcium channels. The molecular basis of the interaction of Sorcin with RyR2 and RyR3 is demonstrated by SPR. Sorcin also interacts with other ER proteins as SERCA2 and Sigma-1 receptor in a calcium-dependent fashion. We also show that Sorcin regulates ER calcium transients: Sorcin increases the velocity of ER calcium uptake (increasing SERCA activity). The data presented here demonstrate that Sorcin may represent both a novel early marker of neurodegenerative diseases and a response to cellular stress dependent on neurodegeneration.
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http://dx.doi.org/10.1038/s41419-020-03063-yDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7566454PMC
October 2020

A multi-dimensional analysis of genotype-phenotype discordance in malignant hyperthermia susceptibility.

Br J Anaesth 2020 12 27;125(6):995-1001. Epub 2020 Aug 27.

Malignant Hyperthermia Investigation Unit, Department of Anesthesia, University Health Network, University of Toronto, Toronto, ON, Canada. Electronic address:

Background: Malignant hyperthermia (MH) susceptibility is an inherited condition, diagnosed either by the presence of a pathogenic genetic variant or by in vitro caffeine-halothane contracture testing. Through a multi-dimensional approach, we describe the implications of discordance between genetic and in vitro test results in a patient with a family history of possible MH.

Methods: The patient, whose brother had a possible MH reaction, underwent the caffeine-halothane contracture test (CHCT) according to the North American MH Group protocol. Screening of the complete RYR1 and CACNA1S transcripts was done using Sanger sequencing. Additional functional analyses included skinned myofibre calcium-induced calcium release sensitivity, calcium signalling assays in cultured myotubes, and in silico evaluation of the effect of any genetic variants on their chemical environment.

Results: The patient's CHCT result was negative but she carried an RYR1 variant c.1209C>G, p.Ile403Met, that is listed as pathogenic by the European Malignant Hyperthermia Group. Functional tests indicated a gain-of-function effect with a weak impact, and the variant was predicted to affect the folding stability of the 3D structure of the RyR1 protein. Based on American College of Medical Genetics and Genomics/Association of Molecular Pathology guidelines, this variant would be characterised as a variant of uncertain significance.

Conclusions: Available data do not confirm or exclude an increased risk of MH for this patient. Further research is needed to correlate RyR1 functional assays, including the current gold standard testing for MH susceptibility, with clinical phenotypes. The pathogenicity of genetic variants associated with MH susceptibility should be re-evaluated.
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http://dx.doi.org/10.1016/j.bja.2020.07.042DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7729844PMC
December 2020

Interleukin-10 and Small Molecule SHIP1 Allosteric Regulators Trigger Anti-inflammatory Effects through SHIP1/STAT3 Complexes.

iScience 2020 Aug 2;23(8):101433. Epub 2020 Aug 2.

Immunity and Infection Research Centre, Vancouver Coastal Health Research Institute, Vancouver, BC V6H 3Z6, Canada; Department of Surgery, University of British Columbia, Vancouver, Canada; Department of Biochemistry and Molecular Biology, University of British Columbia, Vancouver, Canada. Electronic address:

The anti-inflammatory actions of interleukin-10 (IL10) are thought to be mediated primarily by the STAT3 transcription factor, but pro-inflammatory cytokines such as interleukin-6 (IL6) also act through STAT3. We now report that IL10, but not IL6 signaling, induces formation of a complex between STAT3 and the inositol polyphosphate-5-phosphatase SHIP1 in macrophages. Both SHIP1 and STAT3 translocate to the nucleus in macrophages. Remarkably, sesquiterpenes of the Pelorol family, which we previously described as allosteric activators of SHIP1 phosphatase activity, could induce SHIP1/STAT3 complex formation in cells and mimic the anti-inflammatory action of IL10 in a mouse model of colitis. Using crystallography and docking studies we identified a drug-binding pocket in SHIP1. Our studies reveal new mechanisms of action for both STAT3 and SHIP1 and provide a rationale for use of allosteric SHIP1-activating compounds, which mimic the beneficial anti-inflammatory actions of IL10. VIDEO ABSTRACT.
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http://dx.doi.org/10.1016/j.isci.2020.101433DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7452241PMC
August 2020

Structural basis for diamide modulation of ryanodine receptor.

Nat Chem Biol 2020 11 17;16(11):1246-1254. Epub 2020 Aug 17.

Tianjin Key Laboratory for Modern Drug Delivery & High-Efficiency, Collaborative Innovation Center of Chemical Science and Engineering, School of Pharmaceutical Science and Technology, Tianjin University, Tianjin, China.

The diamide insecticide class is one of the top-selling insecticides globally. They are used to control a wide range of pests by targeting their ryanodine receptors (RyRs). Here, we report the highest-resolution cryo-electron microscopy (cryo-EM) structure of RyR1 in the open state, in complex with the anthranilic diamide chlorantraniliprole (CHL). The 3.2-Å local resolution map facilitates unambiguous assignment of the CHL binding site. The molecule induces a conformational change by affecting the S4-S5 linker, triggering channel opening. The binding site is further corroborated by mutagenesis data, which reveal how diamide insecticides are selective to the Lepidoptera group of insects over honeybee or mammalian RyRs. Our data reveal that several pests have developed resistance via two mechanisms, steric hindrance and loss of contact. Our results provide a foundation for the development of highly selective pesticides aimed at overcoming resistance and therapeutic molecules to treat human myopathies.
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http://dx.doi.org/10.1038/s41589-020-0627-5DOI Listing
November 2020

Multiple Sequence Variants in STAC3 Affect Interactions with Ca1.1 and Excitation-Contraction Coupling.

Structure 2020 08 2;28(8):922-932.e5. Epub 2020 Jun 2.

Department of Biochemistry and Molecular Biology, University of British Columbia, Vancouver, BC V6T 1Z3, Canada. Electronic address:

STAC3 is a soluble protein essential for skeletal muscle excitation-contraction (EC) coupling. Through its tandem SH3 domains, it interacts with the cytosolic II-III loop of the skeletal muscle voltage-gated calcium channel. STAC3 is the target for a mutation (W284S) that causes Native American myopathy, but multiple other sequence variants have been reported. Here, we report a crystal structure of the human STAC3 tandem SH3 domains. We analyzed the effect of five disease-associated variants, spread over both SH3 domains, on their ability to bind to the Ca1.1 II-III loop and on muscle EC coupling. In addition to W284S, we find the F295L and K329N variants to affect both binding and EC coupling. The ability of the K329N variant, located in the second SH3 domain, to affect the interaction highlights the importance of both SH3 domains in association with Ca1.1. Our results suggest that multiple STAC3 variants may cause myopathy.
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http://dx.doi.org/10.1016/j.str.2020.05.005DOI Listing
August 2020

The arrhythmogenic N53I variant subtly changes the structure and dynamics in the calmodulin N-terminal domain, altering its interaction with the cardiac ryanodine receptor.

J Biol Chem 2020 05 21;295(22):7620-7634. Epub 2020 Apr 21.

Aalborg University, Department of Chemistry and Bioscience, 9220 Aalborg, Denmark

Mutations in the genes encoding the highly conserved Ca-sensing protein calmodulin (CaM) cause severe cardiac arrhythmias, including catecholaminergic polymorphic ventricular tachycardia or long QT syndrome and sudden cardiac death. Most of the identified arrhythmogenic mutations reside in the C-terminal domain of CaM and mostly affect Ca-coordinating residues. One exception is the catecholaminergic polymorphic ventricular tachycardia-causing N53I substitution, which resides in the N-terminal domain (N-domain). It does not affect Ca coordination and has only a minor impact on binding affinity toward Ca and on other biophysical properties. Nevertheless, the N53I substitution dramatically affects CaM's ability to reduce the open probability of the cardiac ryanodine receptor (RyR2) while having no effect on the regulation of the plasmalemmal voltage-gated Ca channel, Ca1.2. To gain more insight into the molecular disease mechanism of this mutant, we used NMR to investigate the structures and dynamics of both apo- and Ca-bound CaM-N53I in solution. We also solved the crystal structures of WT and N53I CaM in complex with the primary calmodulin-binding domain (CaMBD2) from RyR2 at 1.84-2.13 Å resolutions. We found that all structures of the arrhythmogenic CaM-N53I variant are highly similar to those of WT CaM. However, we noted that the N53I substitution exposes an additional hydrophobic surface and that the intramolecular dynamics of the protein are significantly altered such that they destabilize the CaM N-domain. We conclude that the N53I-induced changes alter the interaction of the CaM N-domain with RyR2 and thereby likely cause the arrhythmogenic phenotype of this mutation.
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http://dx.doi.org/10.1074/jbc.RA120.013430DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7261784PMC
May 2020

Synergy between Cell Surface Glycosidases and Glycan-Binding Proteins Dictates the Utilization of Specific Beta(1,3)-Glucans by Human Gut .

mBio 2020 04 7;11(2). Epub 2020 Apr 7.

Michael Smith Laboratories, University of British Columbia, Vancouver, British Columbia, Canada

The human gut microbiota (HGM) has far-reaching impacts on human health and nutrition, which are fueled primarily by the metabolism of otherwise indigestible complex carbohydrates commonly known as dietary fiber. However, the molecular basis of the ability of individual taxa of the HGM to address specific dietary glycan structures remains largely unclear. In particular, the utilization of β(1,3)-glucans, which are widespread in the human diet as yeast, seaweed, and plant cell walls, had not previously been resolved. Through a systems-based approach, here we show that the symbiont deploys a single, exemplar polysaccharide utilization locus (PUL) to access yeast β(1,3)-glucan, brown seaweed β(1,3)-glucan (laminarin), and cereal mixed-linkage β(1,3)/β(1,4)-glucan. Combined biochemical, enzymatic, and structural analysis of PUL-encoded glycoside hydrolases (GHs) and surface glycan-binding proteins (SGBPs) illuminates a concerted molecular system by which recognizes and saccharifies these distinct β-glucans. Strikingly, the functional characterization of homologous β(1,3)-glucan utilization loci (1,3GUL) in other further demonstrated that the ability of individual taxa to utilize β(1,3)-glucan variants and/or β(1,3)/β(1,4)-glucans arises combinatorially from the individual specificities of SGBPs and GHs at the cell surface, which feed corresponding signals to periplasmic hybrid two-component sensors (HTCSs) via TonB-dependent transporters (TBDTs). These data reveal the importance of cooperativity in the adaptive evolution of GH and SGBP cohorts to address individual polysaccharide structures. We anticipate that this fine-grained knowledge of PUL function will inform metabolic network analysis and proactive manipulation of the HGM. Indeed, a survey of 2,441 public human metagenomes revealed the international, yet individual-specific, distribution of each 1,3GUL. are a dominant phylum of the human gut microbiota (HGM) that target otherwise indigestible dietary fiber with an arsenal of polysaccharide utilization loci (PULs), each of which is dedicated to the utilization of a specific complex carbohydrate. Here, we provide novel insight into this paradigm through functional characterization of homologous PULs from three autochthonous species, which target the family of dietary β(1,3)-glucans. Through detailed biochemical and protein structural analysis, we observed an unexpected diversity in the substrate specificity of PUL glycosidases and glycan-binding proteins with regard to β(1,3)-glucan linkage and branching patterns. In combination, these individual enzyme and protein specificities support taxon-specific growth on individual β(1,3)-glucans. This detailed metabolic insight, together with a comprehensive survey of individual 1,3GULs across human populations, further expands the fundamental roadmap of the HGM, with potential application to the future development of microbial intervention therapies.
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http://dx.doi.org/10.1128/mBio.00095-20DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7157763PMC
April 2020

A rare CACNA1H variant associated with amyotrophic lateral sclerosis causes complete loss of Ca3.2 T-type channel activity.

Mol Brain 2020 03 6;13(1):33. Epub 2020 Mar 6.

Institute of Organic Chemistry and Biochemistry, Czech Academy of Sciences, Flemingovo nam 2, 16610, Prague, Czech Republic.

Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disorder characterized by the progressive loss of cortical, brain stem and spinal motor neurons that leads to muscle weakness and death. A previous study implicated CACNA1H encoding for Ca3.2 calcium channels as a susceptibility gene in ALS. In the present study, two heterozygous CACNA1H variants were identified by whole genome sequencing in a small cohort of ALS patients. These variants were functionally characterized using patch clamp electrophysiology, biochemistry assays, and molecular modeling. A previously unreported c.454GTAC > G variant produced an inframe deletion of a highly conserved isoleucine residue in Ca3.2 (p.ΔI153) and caused a complete loss-of-function of the channel, with an additional dominant-negative effect on the wild-type channel when expressed in trans. In contrast, the c.3629C > T variant caused a missense substitution of a proline with a leucine (p.P1210L) and produced a comparatively mild alteration of Ca3.2 channel activity. The newly identified ΔI153 variant is the first to be reported to cause a complete loss of Ca3.2 channel function. These findings add to the notion that loss-of-function of Ca3.2 channels associated with rare CACNA1H variants may be risk factors in the complex etiology of ALS.
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http://dx.doi.org/10.1186/s13041-020-00577-6DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7060640PMC
March 2020

Arrhythmia mutations in calmodulin can disrupt cooperativity of Ca binding and cause misfolding.

J Physiol 2020 03 18;598(6):1169-1186. Epub 2020 Feb 18.

Department of Biochemistry and Molecular Biology, Life Sciences Institute, University of British Columbia, V6T 1Z3 Vancouver, BC, Canada.

Key Points: Mutations in the calmodulin protein (CaM) are associated with arrhythmia syndromes. This study focuses on understanding the structural characteristics of CaM disease mutants and their interactions with the voltage-gated calcium channel Ca 1.2. Arrhythmia mutations in CaM can lead to loss of Ca binding, uncoupling of Ca binding cooperativity, misfolding of the EF-hands and altered affinity for the calcium channel. These results help us to understand how different CaM mutants have distinct effects on structure and interactions with protein targets to cause disease.

Abstract: Calmodulinopathies are life-threatening arrhythmia syndromes that arise from mutations in calmodulin (CaM), a calcium sensing protein whose sequence is completely conserved across all vertebrates. These mutations have been shown to interfere with the function of cardiac ion channels, including the voltage-gated Ca channel Ca 1.2 and the ryanodine receptor (RyR2), in a mutation-specific manner. The ability of different CaM disease mutations to discriminate between these channels has been enigmatic. We present crystal structures of several C-terminal lobe mutants and an N-terminal lobe mutant in complex with the Ca 1.2 IQ domain, in conjunction with binding assays and complementary structural biology techniques. One mutation (D130G) causes a pathological conformation, with complete separation of EF-hands within the C-lobe and loss of Ca binding in EF-hand 4. Another variant (Q136P) has severely reduced affinity for the IQ domain, and shows changes in the CD spectra under Ca -saturating conditions when unbound to the IQ domain. Ca binding to a pair of EF-hands normally proceeds with very high cooperativity, but we found that N98S CaM can adopt different conformations with either one or two Ca ions bound to the C-lobe, possibly disrupting the cooperativity. An N-lobe variant (N54I), which causes severe stress-induced arrhythmia, does not show any major changes in complex with the IQ domain, providing a structural basis for why this mutant does not affect function of Ca 1.2. These findings show that different CaM mutants have distinct effects on both the CaM structure and interactions with protein targets, and act via distinct pathological mechanisms to cause disease.
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http://dx.doi.org/10.1113/JP279307DOI Listing
March 2020

Cardiac arrest in a mother and daughter and the identification of a novel RYR2 variant, predisposing to low penetrant catecholaminergic polymorphic ventricular tachycardia in a four-generation Canadian family.

Mol Genet Genomic Med 2020 04 28;8(4):e1151. Epub 2020 Jan 28.

Division of Medical Genetics, Island Health, Victoria, BC, Canada.

Background: Catecholaminergic polymorphic ventricular tachycardia (CPVT) is a rare inherited arrhythmia syndrome characterized by adrenergically driven ventricular arrhythmia predominantly caused by pathogenic variants in the cardiac ryanodine receptor (RyR2). We describe a novel variant associated with cardiac arrest in a mother and daughter.

Methods: Initial sequencing of the RYR2 gene identified a novel variant (c.527G > T, p.R176L) in the index case (the mother), and her daughter. Structural analysis demonstrated the variant was located within the N-terminal domain of RyR2, likely leading to a gain-of-function effect facilitating enhanced calcium ion release. Four generation cascade genetic and clinical screening was carried out.

Results: Thirty-eight p.R176L variant carriers were identified of 94 family members with genetic testing, and 108 family members had clinical evaluations. Twelve carriers were symptomatic with previous syncope and 2 additional survivors of cardiac arrest were identified. Thirty-two had clinical features suggestive of CPVT. Of 52 noncarriers, 11 had experienced previous syncope with none exhibiting any clinical features of CPVT. A documented arrhythmic event rate of 2.89/1000 person-years across all carriers was calculated.

Conclusion: The substantial variability in phenotype and the lower than previously reported penetrance is illustrative of the importance of exploring family variants beyond first-degree relatives.
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http://dx.doi.org/10.1002/mgg3.1151DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7196448PMC
April 2020

Nanodisc technology facilitates identification of monoclonal antibodies targeting multi-pass membrane proteins.

Sci Rep 2020 01 24;10(1):1130. Epub 2020 Jan 24.

Amgen Research, Biologic Discovery, Burnaby, BC, Canada.

Multi-pass membrane proteins are important targets of biologic medicines. Given the inherent difficulties in working with membrane proteins, we sought to investigate the utility of membrane scaffold protein nanodiscs as a means of solubilizing membrane proteins to aid antibody discovery. Using a model multi-pass membrane protein, we demonstrate how incorporation of a multi-pass membrane protein into nanodiscs can be used in flow cytometry to identify antigen-specific hybridoma. The use of target protein-loaded nanodiscs to sort individual hybridoma early in the screening process can reduce the time required to identify antibodies against multi-pass membrane proteins.
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http://dx.doi.org/10.1038/s41598-020-58002-wDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6981118PMC
January 2020

Cardiac ryanodine receptor distribution is dynamic and changed by auxiliary proteins and post-translational modification.

Elife 2020 Jan 9;9. Epub 2020 Jan 9.

Department of Cellular and Physiological Sciences, University of British Columbia, Vancouver, Canada.

The effects of the immunophilins, FKBP12 and FKBP12.6, and phosphorylation on type II ryanodine receptor (RyR2) arrangement and function were examined using correlation microscopy (line scan confocal imaging of Ca sparks and dual-tilt electron tomography) and dSTORM imaging of permeabilized Wistar rat ventricular myocytes. Saturating concentrations (10 µmol/L) of either FKBP12 or 12.6 significantly reduced the frequency, spread, amplitude and Ca spark mass relative to control, while the tomograms revealed both proteins shifted the tetramers into a largely side-by-side configuration. Phosphorylation of immunophilin-saturated RyR2 resulted in structural and functional changes largely comparable to phosphorylation alone. dSTORM images of myocyte surfaces demonstrated that both FKBP12 and 12.6 significantly reduced RyR2 cluster sizes, while phosphorylation, even of immunophilin-saturated RyR2, increased them. We conclude that both RyR2 cluster size and the arrangement of tetramers within clusters is dynamic and respond to changes in the cellular environment. Further, these changes affect Ca spark formation.
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http://dx.doi.org/10.7554/eLife.51602DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6994221PMC
January 2020

Molecular interactions between sex hormone-binding globulin and nonsteroidal ligands that enhance androgen activity.

J Biol Chem 2020 01 18;295(5):1202-1211. Epub 2019 Dec 18.

Department of Cellular & Physiological Sciences, The University of British Columbia, Vancouver, BC Canada V6T 1Z4

Sex hormone-binding globulin (SHBG) determines the equilibrium between free and protein-bound androgens and estrogens in the blood and regulates their access to target tissues. Using crystallographic approaches and radiolabeled competitive binding-capacity assays, we report here how two nonsteroidal compounds bind to human SHBG, and how they influence androgen activity in cell culture. We found that one of these compounds, (-)3,4-divanillyltetrahydrofuran (DVT), present in stinging nettle root extracts and used as a nutraceutical, binds SHBG with relatively low affinity. By contrast, a synthetic compound, 3-(1H-imidazol-1-ylmethyl)-2phenyl-1H-indole (IPI), bound SHBG with an affinity similar to that of testosterone and estradiol. Crystal structures of SHBG in complex with DVT or IPI at 1.71-1.80 Å resolutions revealed their unique orientations in the SHBG ligand-binding pocket and suggested opportunities for the design of other nonsteroidal ligands of SHBG. As observed for estradiol but not testosterone, IPI binding to SHBG was reduced by ∼20-fold in the presence of zinc, whereas DVT binding was almost completely lost. Estradiol-dependent fibulin-2 interactions with SHBG similarly occurred for IPI-bound SHBG, but not with DVT-bound SHBG. Both DVT and IPI increased the activity of testosterone in a cell culture androgen reporter system by competitively displacing testosterone from SHBG. These findings indicate how nonsteroidal ligands of SHBG maybe designed to modulate the bioavailability of sex steroids.
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http://dx.doi.org/10.1074/jbc.RA119.011051DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6996888PMC
January 2020

Reply to Pitt and Lee: Occupancies of Ca in complexes of calmodulin with voltage-gated sodium channels.

Proc Natl Acad Sci U S A 2019 Dec 3. Epub 2019 Dec 3.

Department of Biochemistry and Molecular Biology, Life Sciences Institute, University of British Columbia, Vancouver, BC V6T 1Z3, Canada.

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http://dx.doi.org/10.1073/pnas.1918159116DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6936576PMC
December 2019

Slaying a giant: Structures of calmodulin and protein kinase a bound to the cardiac ryanodine receptor.

Cell Calcium 2019 11 6;83:102079. Epub 2019 Sep 6.

University of British Columbia, Life Sciences Institute, Department of Biochemistry and Molecular Biology, 2350 Health Sciences Mall, V6T 1Z3 Vancouver, BC, Canada. Electronic address:

Ryanodine Receptors are Ca release channels expressed in the Endoplasmic and Sarcoplasmic Reticulum membranes. Gong et al [1] reported cryo-EM structures of the cardiac RyR2 complexed to Calmodulin, which can downregulate channel opening. Haji-Ghassemi et al [2] report crystal structures of an RyR2 domain with PKA, a kinase promoting channel opening.
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http://dx.doi.org/10.1016/j.ceca.2019.102079DOI Listing
November 2019

Binding and structural asymmetry governs ligand sensitivity in a cyclic nucleotide-gated ion channel.

J Gen Physiol 2019 10 3;151(10):1190-1212. Epub 2019 Sep 3.

Department of Cellular and Physiological Sciences, University of British Columbia, Vancouver, BC, Canada.

Hyperpolarization-activated cyclic nucleotide-gated (HCN) channels open more easily when cAMP or cGMP bind to a domain in the intracellular C-terminus in each of four identical subunits. How sensitivity of the channels to these ligands is determined is not well understood. Here, we apply a mathematical model, which incorporates negative cooperativity, to gating and mutagenesis data available in the literature and combine the results with binding data collected using isothermal titration calorimetry. This model recapitulates the concentration-response data for the effects of cAMP and cGMP on wild-type HCN2 channel opening and, remarkably, predicts the concentration-response data for a subset of mutants with single-point amino acid substitutions in the binding site. Our results suggest that ligand sensitivity is determined by negative cooperativity and asymmetric effects on structure and channel opening, which are tuned by ligand-specific interactions and residues within the binding site.
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http://dx.doi.org/10.1085/jgp.201812162DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6785730PMC
October 2019

Type 8 long QT syndrome: pathogenic variants in CACNA1C-encoded Cav1.2 cluster in STAC protein binding site.

Europace 2019 Nov;21(11):1725-1732

Division of Cardiology, University of British Columbia, 1033 Davie St., Rm 211, Vancouver, BC, Canada.

Aims: Pathogenic gain-of-function variants in CACAN1C cause type-8 long QT syndrome (LQT8). We sought to describe the electrocardiographic features in LQT8 and utilize molecular modelling to gain mechanistic insights into its genetic culprits.

Methods And Results: Rare variants in CACNA1C were identified from genetic testing laboratories. Treating physicians provided clinical information. Variant pathogenicity was independently assessed according to recent guidelines. Pathogenic (P) and likely pathogenic (LP) variants were mapped onto a 3D modelled structure of the Cav1.2 protein. Nine P/LP variants, identified in 23 patients from 19 families with non-syndromic LQTS were identified. Six variants, found in 79% of families, clustered to a 4-residue section in the cytosolic II-III loop region which forms a region capable of binding STAC SH3 domains. Therefore, variants may affect binding of SH3-domain containing proteins. Arrhythmic events occurred in similar proportions of patients with II-III loop variants and with other P/LP variants (53% vs. 48%, P = 0.41) despite shorter QTc intervals (477 ± 31 ms vs. 515 ± 37 ms, P = 0.03). A history of sudden death was reported only in families with II-III loop variants (60% vs. 0%, P = 0.03). The predominant T-wave morphology was a late peaking T wave with a steep descending limb. Exercise testing demonstrated QTc prolongation on standing and at 4 min recovery after exercise.

Conclusion: The majority of P/LP variants in patients with CACNA1C-mediated LQT8 cluster in an SH3-binding domain of the cytosolic II-III loop. This represents a 'mutation hotspot' in LQT8. A late-peaking T wave with a steep descending limb and QT prolongation on exercise are commonly seen.
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http://dx.doi.org/10.1093/europace/euz215DOI Listing
November 2019

The Cardiac Ryanodine Receptor Phosphorylation Hotspot Embraces PKA in a Phosphorylation-Dependent Manner.

Mol Cell 2019 07 8;75(1):39-52.e4. Epub 2019 May 8.

Department of Biochemistry and Molecular Biology, Life Sciences Institute, University of British Columbia, Vancouver, BC V6T 1Z3, Canada. Electronic address:

Ryanodine receptors (RyRs) are intracellular Ca release channels controlling essential cellular functions. RyRs are targeted by cyclic AMP (cAMP)-dependent protein kinase A (PKA), a controversial regulation implicated in disorders ranging from heart failure to Alzheimer's. Using crystal structures, we show that the phosphorylation hotspot domain of RyR2 embraces the PKA catalytic subunit, with an extensive interface not seen in PKA complexes with peptides. We trapped an intermediary open-form PKA bound to the RyR2 domain and an ATP analog, showing that PKA can engage substrates in an open form. Phosphomimetics or prior phosphorylation at nearby sites in RyR2 either enhance or reduce the activity of PKA. Finally, we show that a phosphomimetic at S2813, a well-known target site for calmodulin-dependent kinase II, induces the formation of an alpha helix in the phosphorylation domain, resulting in increased interactions and PKA activity. This shows that the different phosphorylation sites in RyR2 are not independent.
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http://dx.doi.org/10.1016/j.molcel.2019.04.019DOI Listing
July 2019

Crystal structures of Ca-calmodulin bound to Na C-terminal regions suggest role for EF-hand domain in binding and inactivation.

Proc Natl Acad Sci U S A 2019 05 9;116(22):10763-10772. Epub 2019 May 9.

Department of Biochemistry and Molecular Biology, Life Sciences Institute, The University of British Columbia, Vancouver, BC, Canada V6T 1Z3

Voltage-gated sodium (Na) and calcium channels (Ca) form targets for calmodulin (CaM), which affects channel inactivation properties. A major interaction site for CaM resides in the C-terminal (CT) region, consisting of an IQ domain downstream of an EF-hand domain. We present a crystal structure of fully Ca-occupied CaM, bound to the CT of Na1.5. The structure shows that the C-terminal lobe binds to a site ∼90° rotated relative to a previous site reported for an apoCaM complex with the Na1.5 CT and for ternary complexes containing fibroblast growth factor homologous factors (FHF). We show that the binding of FHFs forces the EF-hand domain in a conformation that does not allow binding of the Ca-occupied C-lobe of CaM. These observations highlight the central role of the EF-hand domain in modulating the binding mode of CaM. The binding sites for Ca-free and Ca-occupied CaM contain targets for mutations linked to long-QT syndrome, a type of inherited arrhythmia. The related Na1.4 channel has been shown to undergo Ca-dependent inactivation (CDI) akin to Cas. We present a crystal structure of Ca/CaM bound to the Na1.4 IQ domain, which shows a binding mode that would clash with the EF-hand domain. We postulate the relative reorientation of the EF-hand domain and the IQ domain as a possible conformational switch that underlies CDI.
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http://dx.doi.org/10.1073/pnas.1818618116DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6561220PMC
May 2019

Surface glycan-binding proteins are essential for cereal beta-glucan utilization by the human gut symbiont Bacteroides ovatus.

Cell Mol Life Sci 2019 Nov 6;76(21):4319-4340. Epub 2019 May 6.

Michael Smith Laboratories, University of British Columbia, 2185 East Mall, Vancouver, BC, V6T 1Z4, Canada.

The human gut microbiota, which underpins nutrition and systemic health, is compositionally sensitive to the availability of complex carbohydrates in the diet. The Bacteroidetes comprise a dominant phylum in the human gut microbiota whose members thrive on dietary and endogenous glycans by employing a diversity of highly specific, multi-gene polysaccharide utilization loci (PUL), which encode a variety of carbohydrases, transporters, and sensor/regulators. PULs invariably also encode surface glycan-binding proteins (SGBPs) that play a central role in saccharide capture at the outer membrane. Here, we present combined biophysical, structural, and in vivo characterization of the two SGBPs encoded by the Bacteroides ovatus mixed-linkage β-glucan utilization locus (MLGUL), thereby elucidating their key roles in the metabolism of this ubiquitous dietary cereal polysaccharide. In particular, molecular insight gained through several crystallographic complexes of SGBP-A and SGBP-B with oligosaccharides reveals that unique shape complementarity of binding platforms underpins specificity for the kinked MLG backbone vis-à-vis linear β-glucans. Reverse-genetic analysis revealed that both the presence and binding ability of the SusD homolog BoSGBP-A are essential for growth on MLG, whereas the divergent, multi-domain BoSGBP-B is dispensable but may assist in oligosaccharide scavenging from the environment. The synthesis of these data illuminates the critical role SGBPs play in concert with other MLGUL components, reveals new structure-function relationships among SGBPs, and provides fundamental knowledge to inform future (meta)genomic, biochemical, and microbiological analyses of the human gut microbiota.
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http://dx.doi.org/10.1007/s00018-019-03115-3DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6810844PMC
November 2019

In vitro analyses of suspected arrhythmogenic thin filament variants as a cause of sudden cardiac death in infants.

Proc Natl Acad Sci U S A 2019 04 18;116(14):6969-6974. Epub 2019 Mar 18.

Molecular Cardiac Physiology Group, Simon Fraser University, Burnaby, BC V5A 1S6, Canada;

Sudden unexpected death of an infant (SUDI) is a devastating occurrence for families. To investigate the genetic pathogenesis of SUDI, we sequenced >70 genes from 191 autopsy-negative SUDI victims. Ten infants sharing a previously unknown variant in troponin I (TnI) were identified. The mutation ( R37C) is in the fetal/neonatal paralog of TnI, a gene thought to be expressed in the heart up to the first 24 months of life. Using phylogenetic analysis and molecular dynamics simulations, it was determined that arginine at residue 37 in may play a critical functional role, suggesting that the variant may be pathogenic. We investigated the biophysical properties of the R37C mutation in human reconstituted thin filaments (RTFs) using fluorometry. RTFs reconstituted with the mutant R37C TnI exhibited reduced Ca-binding sensitivity due to an increased Ca off-rate constant. Furthermore, we generated R37C mutants in human induced pluripotent stem cell derived cardiomyocytes (hiPSC-CMs) using CRISPR-Cas9. In monolayers of hiPSC-CMs, we simultaneously monitored voltage and Ca transients through optical mapping and compared them to their isogenic controls. We observed normal intrinsic beating patterns under control conditions in R37C at stimulation frequencies of 55 beats/min (bpm), but these cells showed no restitution with increased stimulation frequency to 65 bpm and exhibited alternans at >75 bpm. The WT hiPSC-CMs did not exhibit any sign of arrhythmogenicity even at stimulation frequencies of 120 bpm. The approach used in this study provides critical physiological and mechanistic bases to investigate sarcomeric mutations in the pathogenesis of SUDI.
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http://dx.doi.org/10.1073/pnas.1819023116DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6452669PMC
April 2019

Cardiac hypertrophy and arrhythmia in mice induced by a mutation in ryanodine receptor 2.

JCI Insight 2019 03 5;5. Epub 2019 Mar 5.

Department of Medicine, Division of Cardiovascular Medicine, and Cardiovascular Research Center, University of Wisconsin-Madison School of Medicine and Public Health, Madison, Wisconsin, USA.

Hypertrophic cardiomyopathy (HCM) is triggered mainly by mutations in genes encoding sarcomeric proteins, but a significant proportion of patients lack a genetic diagnosis. We identified a novel mutation in the ryanodine receptor 2, RyR2-P1124L, in a patient from a genotype-negative HCM cohort. The aim of this study was to determine whether RyR2-P1124L triggers functional and structural alterations in isolated RyR2 channels and whole hearts. We found that P1124L induces significant conformational changes in the SPRY2 domain of RyR2. Recombinant RyR2-P1124L channels displayed a cytosolic loss-of-function phenotype, which contrasted with a higher sensitivity to luminal [Ca2+], indicating a luminal gain-of-function. Homozygous mice for RyR2-P1124L showed mild cardiac hypertrophy, similar to the human patient. This phenotype, evident at 1 yr of age, was accompanied by an increase in the expression of calmodulin (CaM). P1124L mice also showed higher susceptibility to arrhythmia at 8 mo of age, before the onset of hypertrophy. RyR2-P1124L has a distinct cytosolic loss-of-function and a luminal gain-of-function phenotype. This bifunctionally-divergent behavior triggers arrhythmias and structural cardiac remodeling, and involves overexpression of calmodulin as a potential hypertrophic mediator. This study is relevant to continue elucidating the possible causes of genotype-negative HCM and the role of RyR2 in cardiac hypertrophy.
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http://dx.doi.org/10.1172/jci.insight.126544DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6483635PMC
March 2019

Subtle Changes in the Combining Site of the Chlamydiaceae-Specific mAb S25-23 Increase the Antibody-Carbohydrate Binding Affinity by an Order of Magnitude.

Biochemistry 2019 02 10;58(6):714-726. Epub 2019 Jan 10.

Department of Biochemistry and Microbiology , University of Victoria , P.O. Box 3055 STN CSC, Victoria , British Columbia , Canada V8P 3P6.

Murine antibodies S25-23, S25-26, and S25-5 derive from a common germ-line origin, and all bind the Chlamydiaceae family-specific epitope αKdo(2→8)αKdo(2→4)αKdo (where Kdo is 3-deoxy-α-d- manno-oct-2-ulosonic acid) with high affinity and specificity. These antibodies recognize the entire trisaccharide antigen in a linkage-dependent manner via a groove composed largely of germ-line residues. Despite sharing identical heavy and light chain genes, S25-23 binds the family-specific epitope with nanomolar affinity, which is an order of magnitude higher than that of S25-26, while S25-5 displays an affinity between those of S25-23 and S25-26. We determined the high-resolution crystal structures of S25-23 and S25-5 antigen binding fragments in complex with a pentasaccharide derived from the LPS of Chlamydia and measured the affinity of S25-5 for chlamydial LPS antigens using isothermal titration microcalorimetry. The 1.75 Å resolution structure of S25-23 shows how subtle conservative mutations Arg(L)-27E to lysine and Ser(H)-56 to threonine lead to an order of magnitude increase in affinity. Importantly, comparison between previous S25-26 structures and the 1.99 and 2.05 Å resolution liganded and unliganded structures of S25-5, respectively, shows how a Ser(L)-27E mutation results in an intermediate affinity due to the reduced enthalpic penalty associated with complex formation that would otherwise be required for arginine in this position. This strategy allows for subtle adjustments in the combining site via affinity maturation that have dramatic consequences for the affinity of an antibody for its antigen.
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http://dx.doi.org/10.1021/acs.biochem.8b00318DOI Listing
February 2019

Ca-dependent calmodulin binding to cardiac ryanodine receptor (RyR2) calmodulin-binding domains.

Biochem J 2019 01 18;476(2):193-209. Epub 2019 Jan 18.

Department of Chemistry and Bioscience, Aalborg University, 9220 Aalborg, Denmark

The Ca sensor calmodulin (CaM) regulates cardiac ryanodine receptor (RyR2)-mediated Ca release from the sarcoplasmic reticulum. CaM inhibits RyR2 in a Ca-dependent manner and aberrant CaM-dependent inhibition results in life-threatening cardiac arrhythmias. However, the molecular details of the CaM-RyR2 interaction remain unclear. Four CaM-binding domains (CaMBD1a, -1b, -2, and -3) in RyR2 have been proposed. Here, we investigated the Ca-dependent interactions between CaM and these CaMBDs by monitoring changes in the fluorescence anisotropy of carboxytetramethylrhodamine (TAMRA)-labeled CaMBD peptides during titration with CaM at a wide range of Ca concentrations. We showed that CaM bound to all four CaMBDs with affinities that increased with Ca concentration. CaM bound to CaMBD2 and -3 with high affinities across all Ca concentrations tested, but bound to CaMBD1a and -1b only at Ca concentrations above 0.2 µM. Binding experiments using individual CaM domains revealed that the CaM C-domain preferentially bound to CaMBD2, and the N-domain to CaMBD3. Moreover, the Ca affinity of the CaM C-domain in complex with CaMBD2 or -3 was so high that these complexes are essentially Ca saturated under resting Ca conditions. Conversely, the N-domain senses Ca exactly in the transition from resting to activating Ca when complexed to either CaMBD2 or -3. Altogether, our results support a binding model where the CaM C-domain is anchored to RyR2 CaMBD2 and saturated with Ca during Ca oscillations, while the CaM N-domain functions as a dynamic Ca sensor that can bridge noncontiguous regions of RyR2 or clamp down onto CaMBD2.
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http://dx.doi.org/10.1042/BCJ20180545DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6340113PMC
January 2019

Catecholaminergic polymorphic ventricular tachycardia patients with multiple genetic variants in the PACES CPVT Registry.

PLoS One 2018 7;13(11):e0205925. Epub 2018 Nov 7.

Departments of Medicine, Pediatrics, and Biochemistry & Molecular Biology, University of British Columbia, Vancouver, BC, Canada.

Background: Catecholaminergic polymorphic ventricular tachycardia (CPVT) is often a life-threatening arrhythmia disorder with variable penetrance and expressivity. Little is known about the incidence or outcomes of CPVT patients with ≥2 variants.

Methods: The phenotypes, genotypes and outcomes of patients in the Pediatric and Congenital Electrophysiology Society CPVT Registry with ≥2 variants in genes linked to CPVT were ascertained. The American College of Medical Genetics & Genomics (ACMG) criteria and structural mapping were used to predict the pathogenicity of variants (3D model of pig RyR2 in open-state).

Results: Among 237 CPVT subjects, 193 (81%) had genetic testing. Fifteen patients (8%) with a median age of 9 years (IQR 5-12) had ≥2 variants. Sudden cardiac arrest occurred in 11 children (73%), although none died during a median follow-up of 4.3 years (IQR 2.5-6.1). Thirteen patients (80%) had at least two RYR2 variants, while the remaining two patients had RYR2 variants plus variants in other CPVT-linked genes. Among all variants identified, re-classification of the commercial laboratory interpretation using ACMG criteria led to the upgrade from variant of unknown significance (VUS) to pathogenic/likely pathogenic (P/LP) for 5 variants, and downgrade from P/LP to VUS for 6 variants. For RYR2 variants, 3D mapping using the RyR2 model suggested that 2 VUS by ACMG criteria were P/LP, while 2 variants were downgraded to likely benign.

Conclusions: This severely affected cohort demonstrates that a minority of CPVT cases are related to ≥2 variants, which may have implications on family-based genetic counselling. While multi-variant CPVT patients were at high-risk for sudden cardiac arrest, there are insufficient data to conclude that this genetic phenomenon has prognostic implications at present. Further research is needed to determine the significance and generalizability of this observation. This study also shows that a rigorous approach to variant re-classification using the ACMG criteria and 3D mapping is important in reaching an accurate diagnosis, especially in the multi-variant population.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0205925PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6221297PMC
April 2019

Arrhythmia mutations in calmodulin cause conformational changes that affect interactions with the cardiac voltage-gated calcium channel.

Proc Natl Acad Sci U S A 2018 11 22;115(45):E10556-E10565. Epub 2018 Oct 22.

Department of Biochemistry and Molecular Biology, University of British Columbia, Vancouver, BC V6T 1Z3, Canada;

Calmodulin (CaM) represents one of the most conserved proteins among eukaryotes and is known to bind and modulate more than a 100 targets. Recently, several disease-associated mutations have been identified in the genes that are causative of severe cardiac arrhythmia syndromes. Although several mutations have been shown to affect the function of various cardiac ion channels, direct structural insights into any CaM disease mutation have been lacking. Here we report a crystallographic and NMR investigation of several disease mutant CaMs, linked to long-QT syndrome, in complex with the IQ domain of the cardiac voltage-gated calcium channel (Ca1.2). Surprisingly, two mutants (D95V, N97I) cause a major distortion of the C-terminal lobe, resulting in a pathological conformation not reported before. These structural changes result in altered interactions with the Ca1.2 IQ domain. Another mutation (N97S) reduces the affinity for Ca by introducing strain in EF hand 3. A fourth mutant (F141L) shows structural changes in the Ca-free state that increase the affinity for the IQ domain. These results thus show that different mechanisms underlie the ability of CaM disease mutations to affect Ca-dependent inactivation of the voltage-gated calcium channel.
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http://dx.doi.org/10.1073/pnas.1808733115DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6233071PMC
November 2018

Calcium Channelopathies: Structural Insights into Disorders of the Muscle Excitation-Contraction Complex.

Annu Rev Genet 2018 11 12;52:373-396. Epub 2018 Sep 12.

Department of Biochemistry and Molecular Biology, University of British Columbia, Vancouver, British Columbia V6T 1Z3, Canada; email:

Ion channels are membrane proteins responsible for the passage of ions down their electrochemical gradients and across biological membranes. In this, they generate and shape action potentials and provide secondary messengers for various signaling pathways. They are often part of larger complexes containing auxiliary subunits and regulatory proteins. Channelopathies arise from mutations in the genes encoding ion channels or their associated proteins. Recent advances in cryo-electron microscopy have resulted in an explosion of ion channel structures in multiple states, generating a wealth of new information on channelopathies. Disease-associated mutations fall into different categories, interfering with ion permeation, protein folding, voltage sensing, ligand and protein binding, and allosteric modulation of channel gating. Prime examples of these are Ca-selective channels expressed in myocytes, for which multiple structures in distinct conformational states have recently been uncovered. We discuss the latest insights into these calcium channelopathies from a structural viewpoint.
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http://dx.doi.org/10.1146/annurev-genet-120417-031311DOI Listing
November 2018