Publications by authors named "Oliver B Clarke"

38 Publications

Physiologically Relevant Free Ca Ion Concentrations Regulate STRA6-Calmodulin Complex Formation via the BP2 Region of STRA6.

J Mol Biol 2021 Sep 27;433(22):167272. Epub 2021 Sep 27.

The Center for Biomolecular Therapeutics (CBT), Department of Biochemistry and Molecular Biology University of Maryland School of Medicine, 108 N. Greene St, Baltimore, MD 21201, USA; The Institute of Bioscience and Biotechnology Research (IBBR), 9600 Gudelsky Dr., Rockville, MD 20850, USA. Electronic address:

The interaction of calmodulin (CaM) with the receptor for retinol uptake, STRA6, involves an α-helix termed BP2 that is located on the intracellular side of this homodimeric transporter (Chen et al., 2016 [1]). In the absence of Ca, NMR data showed that a peptide derived from BP2 bound to the C-terminal lobe (C-lobe) of Mg-bound CaM (CaM). Upon titration of Ca into CaM-BP2, NMR chemical shift perturbations (CSPs) were observed for residues in the C-lobe, including those in the EF-hand Ca-binding domains, EF3 and EF4 (K = 60 ± 7 nM). As higher concentrations of free Ca were achieved, CSPs occurred for residues in the N-terminal lobe (N-lobe) including those in EF1 and EF2 (K = 1000 ± 160 nM). Thermodynamic and kinetic Ca binding studies showed that BP2 addition increased the Ca-binding affinity of CaM and slowed its Ca dissociation rates (k) in both the C- and N-lobe EF-hand domains, respectively. These data are consistent with BP2 binding to the C-lobe of CaM at low free Ca concentrations (<100 nM) like those found at resting intracellular levels. As free Ca levels approach 1000 nM, which is typical inside a cell upon an intracellular Ca-signaling event, BP2 is shown here to interact with both the N- and C-lobes of Ca-loaded CaM (CaM-BP2). Because this structural rearrangement observed for the CaM-BP2 complex occurs as intracellular free Ca concentrations approach those typical of a Ca-signaling event (K = 1000 ± 160 nM), this conformational change could be relevant to vitamin A transport by full-length CaM-STRA6.
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http://dx.doi.org/10.1016/j.jmb.2021.167272DOI Listing
September 2021

High-resolution structure of the membrane-embedded skeletal muscle ryanodine receptor.

Structure 2021 Aug 26. Epub 2021 Aug 26.

Department of Physiology and Cellular Biophysics, Columbia University Vagelos College of Physicians & Surgeons, New York, NY, USA; Clyde & Helen Wu Center for Molecular Cardiology, Columbia University Vagelos College of Physicians & Surgeons, New York, NY, USA. Electronic address:

The type 1 ryanodine receptor (RyR)/calcium release channel on the sarcoplasmic reticulum (SR) is required for skeletal muscle excitation-contraction coupling and is the largest known ion channel, composed of four 565-kDa protomers. Cryogenic electron microscopy (cryo-EM) studies of the RyR have primarily used detergent to solubilize the channel; in the present study, we have used cryo-EM to solve high-resolution structures of the channel in liposomes using a gel-filtration approach with on-column detergent removal to form liposomes and incorporate the channel simultaneously. This allowed us to resolve the structure of the channel in the primed and open states at 3.4 and 4.0 Å, respectively, with a single dataset. This method offers validation for detergent-based structures of the RyR and offers a starting point for utilizing a chemical gradient mimicking the SR, where Ca concentrations are millimolar in the lumen and nanomolar in the cytosol.
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http://dx.doi.org/10.1016/j.str.2021.08.001DOI Listing
August 2021

Gating movements and ion permeation in HCN4 pacemaker channels.

Mol Cell 2021 07 23;81(14):2929-2943.e6. Epub 2021 Jun 23.

Department of Biosciences, University of Milan, Milan, Italy; Institute of Biophysics-Milano, Consiglio Nazionale delle Ricerche, Rome, Italy. Electronic address:

The HCN1-4 channel family is responsible for the hyperpolarization-activated cation current I/I that controls automaticity in cardiac and neuronal pacemaker cells. We present cryoelectron microscopy (cryo-EM) structures of HCN4 in the presence or absence of bound cAMP, displaying the pore domain in closed and open conformations. Analysis of cAMP-bound and -unbound structures sheds light on how ligand-induced transitions in the channel cytosolic portion mediate the effect of cAMP on channel gating and highlights the regulatory role of a Mg coordination site formed between the C-linker and the S4-S5 linker. Comparison of open/closed pore states shows that the cytosolic gate opens through concerted movements of the S5 and S6 transmembrane helices. Furthermore, in combination with molecular dynamics analyses, the open pore structures provide insights into the mechanisms of K/Na permeation. Our results contribute mechanistic understanding on HCN channel gating, cyclic nucleotide-dependent modulation, and ion permeation.
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http://dx.doi.org/10.1016/j.molcel.2021.05.033DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8294335PMC
July 2021

Structural basis of omega-3 fatty acid transport across the blood-brain barrier.

Nature 2021 07 16;595(7866):315-319. Epub 2021 Jun 16.

Department of Physiology and Cellular Biophysics, Columbia University, New York, NY, USA.

Docosahexaenoic acid is an omega-3 fatty acid that is essential for neurological development and function, and it is supplied to the brain and eyes predominantly from dietary sources. This nutrient is transported across the blood-brain and blood-retina barriers in the form of lysophosphatidylcholine by major facilitator superfamily domain containing 2A (MFSD2A) in a Na-dependent manner. Here we present the structure of MFSD2A determined using single-particle cryo-electron microscopy, which reveals twelve transmembrane helices that are separated into two pseudosymmetric domains. The transporter is in an inward-facing conformation and features a large amphipathic cavity that contains the Na-binding site and a bound lysolipid substrate, which we confirmed using native mass spectrometry. Together with our functional analyses and molecular dynamics simulations, this structure reveals details of how MFSD2A interacts with substrates and how Na-dependent conformational changes allow for the release of these substrates into the membrane through a lateral gate. Our work provides insights into the molecular mechanism by which this atypical major facility superfamily transporter mediates the uptake of lysolipids into the brain, and has the potential to aid in the delivery of neurotherapeutic agents.
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http://dx.doi.org/10.1038/s41586-021-03650-9DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8266758PMC
July 2021

Structure and activity of SLAC1 channels for stomatal signaling in leaves.

Proc Natl Acad Sci U S A 2021 May;118(18)

State Key Laboratory of Molecular Developmental Biology, Institute of Genetics and Developmental Biology, Chinese Academy of Sciences, 100101 Beijing, China;

Stomata in leaves regulate gas exchange between the plant and its atmosphere. Various environmental stimuli elicit abscisic acid (ABA); ABA leads to phosphoactivation of slow anion channel 1 (SLAC1); SLAC1 activity reduces turgor pressure in aperture-defining guard cells; and stomatal closure ensues. We used electrophysiology for functional characterizations of SLAC1 (SLAC1) and cryoelectron microscopy (cryo-EM) for structural analysis of SLAC1 (SLAC1), at 2.97-Å resolution. We identified 14 phosphorylation sites in SLAC1 and showed nearly 330-fold channel-activity enhancement with 4 to 6 of these phosphorylated. Seven SLAC1-conserved arginines are poised in SLAC1 for regulatory interaction with the N-terminal extension. This SLAC1 structure has its pores closed, in a basal state, spring loaded by phenylalanyl residues in high-energy conformations. SLAC1 phosphorylation fine-tunes an equilibrium between basal and activated SLAC1 trimers, thereby controlling the degree of stomatal opening.
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http://dx.doi.org/10.1073/pnas.2015151118DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8106318PMC
May 2021

Structural Basis of WLS/Evi-Mediated Wnt Transport and Secretion.

Cell 2021 01 23;184(1):194-206.e14. Epub 2020 Dec 23.

Department of Physiology and Cellular Biophysics, Columbia University Irving Medical Center, New York, NY 10032, USA. Electronic address:

Wnts are evolutionarily conserved ligands that signal at short range to regulate morphogenesis, cell fate, and stem cell renewal. The first and essential steps in Wnt secretion are their O-palmitoleation and subsequent loading onto the dedicated transporter Wntless/evenness interrupted (WLS/Evi). We report the 3.2 Å resolution cryogenic electron microscopy (cryo-EM) structure of palmitoleated human WNT8A in complex with WLS. This is accompanied by biochemical experiments to probe the physiological implications of the observed association. The WLS membrane domain has close structural homology to G protein-coupled receptors (GPCRs). A Wnt hairpin inserts into a conserved hydrophobic cavity in the GPCR-like domain, and the palmitoleate protrudes between two helices into the bilayer. A conformational switch of highly conserved residues on a separate Wnt hairpin might contribute to its transfer to receiving cells. This work provides molecular-level insights into a central mechanism in animal body plan development and stem cell biology.
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http://dx.doi.org/10.1016/j.cell.2020.11.038DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7797000PMC
January 2021

Structure of the Plasmodium falciparum PfSERA5 pseudo-zymogen.

Protein Sci 2020 11 5;29(11):2245-2258. Epub 2020 Oct 5.

Department of Chemistry and Physics, La Trobe Institute for Molecular Science, La Trobe University, Melbourne, Victoria, Australia.

PfSERA5, a significantly abundant protein present within the parasitophorous vacuole (PV) and essential for normal growth during the blood-stage life cycle of the malaria parasite Plasmodium falciparum, displays structural similarity to many other cysteine proteases. However, PfSERA5 does not exhibit any detectable protease activity and therefore the role of the PfSERA5 papain-like domain (PfSERA5E), thought to remain bound to its cognate prodomain, remains unknown. In this study, we present a revised structure of the central PfSERA5E domain at a resolution of 1.2 Å, and the first structure of the "zymogen" of this papain-like domain including its cognate prodomain (PfSERA5PE) to 2.2 Å resolution. PfSERA5PE is somewhat structurally similar to that of other known proenzymes, retaining the conserved overall folding and orientation of the prodomain through, and occluding, the archetypal papain-like catalytic triad "active-site" cleft, in the same reverse direction as conventional prodomains. Our findings are congruent with previously identified structures of PfSERA5E and of similar "zymogens" and provide a foundation for further investigation into the function of PfSERA5.
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http://dx.doi.org/10.1002/pro.3956DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7586913PMC
November 2020

Author Correction: Structure of human GABA receptor in an inactive state.

Nature 2020 Jul;583(7818):E29

Howard Hughes Medical Institute, Department of Pharmacology, University of Texas Southwestern Medical Center, Dallas, TX, USA.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.
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http://dx.doi.org/10.1038/s41586-020-2543-yDOI Listing
July 2020

Cryo-EM structure of arabinosyltransferase EmbB from Mycobacterium smegmatis.

Nat Commun 2020 07 7;11(1):3396. Epub 2020 Jul 7.

Department of Physiology and Cellular Biophysics, Columbia University, New York, NY, 10032, USA.

Arabinosyltransferase B (EmbB) belongs to a family of membrane-bound glycosyltransferases that build the lipidated polysaccharides of the mycobacterial cell envelope, and are targets of anti-tuberculosis drug ethambutol. We present the 3.3 Å resolution single-particle cryo-electron microscopy structure of Mycobacterium smegmatis EmbB, providing insights on substrate binding and reaction mechanism. Mutations that confer ethambutol resistance map mostly around the putative active site, suggesting this to be the location of drug binding.
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http://dx.doi.org/10.1038/s41467-020-17202-8DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7341804PMC
July 2020

Structure of human GABA receptor in an inactive state.

Nature 2020 08 24;584(7820):304-309. Epub 2020 Jun 24.

Howard Hughes Medical Institute, Department of Pharmacology, University of Texas Southwestern Medical Center, Dallas, TX, USA.

The human GABA receptor-a member of the class C family of G-protein-coupled receptors (GPCRs)-mediates inhibitory neurotransmission and has been implicated in epilepsy, pain and addiction. A unique GPCR that is known to require heterodimerization for function, the GABA receptor has two subunits, GABA and GABA, that are structurally homologous but perform distinct and complementary functions. GABA recognizes orthosteric ligands, while GABA couples with G proteins. Each subunit is characterized by an extracellular Venus flytrap (VFT) module, a descending peptide linker, a seven-helix transmembrane domain and a cytoplasmic tail. Although the VFT heterodimer structure has been resolved, the structure of the full-length receptor and its transmembrane signalling mechanism remain unknown. Here we present a near full-length structure of the GABA receptor, captured in an inactive state by cryo-electron microscopy. Our structure reveals several ligands that preassociate with the receptor, including two large endogenous phospholipids that are embedded within the transmembrane domains to maintain receptor integrity and modulate receptor function. We also identify a previously unknown heterodimer interface between transmembrane helices 3 and 5 of both subunits, which serves as a signature of the inactive conformation. A unique 'intersubunit latch' within this transmembrane interface maintains the inactive state, and its disruption leads to constitutive receptor activity.
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http://dx.doi.org/10.1038/s41586-020-2452-0DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7725281PMC
August 2020

A constricted opening in Kir channels does not impede potassium conduction.

Nat Commun 2020 06 15;11(1):3024. Epub 2020 Jun 15.

Structural Biology Division, The Walter and Eliza Hall Institute of Medical Research, Parkville, VIC, 3052, Australia.

The canonical mechanistic model explaining potassium channel gating is of a conformational change that alternately dilates and constricts a collar-like intracellular entrance to the pore. It is based on the premise that K ions maintain a complete hydration shell while passing between the transmembrane cavity and cytosol, which must be accommodated. To put the canonical model to the test, we locked the conformation of a Kir K channel to prevent widening of the narrow collar. Unexpectedly, conduction was unimpaired in the locked channels. In parallel, we employed all-atom molecular dynamics to simulate K ions moving along the conduction pathway between the lower cavity and cytosol. During simulations, the constriction did not significantly widen. Instead, transient loss of some water molecules facilitated K permeation through the collar. The low free energy barrier to partial dehydration in the absence of conformational change indicates Kir channels are not gated by the canonical mechanism.
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http://dx.doi.org/10.1038/s41467-020-16842-0DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7295778PMC
June 2020

Structural and Functional Characterization of Phosphatidylinositol-Phosphate Biosynthesis in Mycobacteria.

J Mol Biol 2020 08 8;432(18):5137-5151. Epub 2020 May 8.

Department of Physiology and Cellular Biophysics, Columbia University, New York, NY 10032, USA. Electronic address:

In mycobacteria, phosphatidylinositol (PI) acts as a common lipid anchor for key components of the cell wall, including the glycolipids phosphatidylinositol mannoside, lipomannan, and lipoarabinomannan. Glycolipids in Mycobacterium tuberculosis, the causative agent of tuberculosis, are important virulence factors that modulate the host immune response. The identity-defining step in PI biosynthesis in prokaryotes, unique to mycobacteria and few other bacterial species, is the reaction between cytidine diphosphate-diacylglycerol and inositol-phosphate to yield phosphatidylinositol-phosphate, the immediate precursor to PI. This reaction is catalyzed by the cytidine diphosphate-alcohol phosphotransferase phosphatidylinositol-phosphate synthase (PIPS), an essential enzyme for mycobacterial viability. Here we present structures of PIPS from Mycobacterium kansasii with and without evidence of donor and acceptor substrate binding obtained using a crystal engineering approach. PIPS from Mycobacterium kansasii is 86% identical to the ortholog from M. tuberculosis and catalytically active. Functional experiments guided by our structural results allowed us to further characterize the molecular determinants of substrate specificity and catalysis in a new mycobacterial species. This work provides a framework to strengthen our understanding of phosphatidylinositol-phosphate biosynthesis in the context of mycobacterial pathogens.
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http://dx.doi.org/10.1016/j.jmb.2020.04.028DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7483940PMC
August 2020

Cryo-EM Structures and Regulation of Arabinofuranosyltransferase AftD from Mycobacteria.

Mol Cell 2020 05 7;78(4):683-699.e11. Epub 2020 May 7.

Department of Physiology and Cellular Biophysics, Columbia University, New York, NY 10032, USA. Electronic address:

Mycobacterium tuberculosis causes tuberculosis, a disease that kills over 1 million people each year. Its cell envelope is a common antibiotic target and has a unique structure due, in part, to two lipidated polysaccharides-arabinogalactan and lipoarabinomannan. Arabinofuranosyltransferase D (AftD) is an essential enzyme involved in assembling these glycolipids. We present the 2.9-Å resolution structure of M. abscessus AftD, determined by single-particle cryo-electron microscopy. AftD has a conserved GT-C glycosyltransferase fold and three carbohydrate-binding modules. Glycan array analysis shows that AftD binds complex arabinose glycans. Additionally, AftD is non-covalently complexed with an acyl carrier protein (ACP). 3.4- and 3.5-Å structures of a mutant with impaired ACP binding reveal a conformational change, suggesting that ACP may regulate AftD function. Mutagenesis experiments using a conditional knockout constructed in M. smegmatis confirm the essentiality of the putative active site and the ACP binding for AftD function.
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http://dx.doi.org/10.1016/j.molcel.2020.04.014DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7263364PMC
May 2020

Sample preparation for structural and functional analyses of the STRA6 receptor for retinol-binding protein.

Methods Enzymol 2020 9;637:95-117. Epub 2020 Apr 9.

Department of Physiology and Cellular Biophysics, Columbia University Irving Medical Center, New York, NY, United States. Electronic address:

STRA6 (stimulated by retinoic acid 6) is a 75kDa polytopic transmembrane protein that facilitates cellular retinol uptake from retinol-binding protein (RBP). Structural characterization of STRA6 from Danio rerio purified in detergent and reconstituted in amphipol A8-35 was achieved by single-particle cryo-electron microscopy (cryo-EM). This provided the first high-resolution snapshot of this protein, showing a novel topology of a tightly assembled homodimer, and an unexpected physiological association with calmodulin in addition to insights into its potential mechanism of function. Specifically, a large hydrophobic cavity in the center of STRA6 linked to the known site of interaction with RBP suggested a route of retinol entry into the cell by diffusion into the membrane through a lateral opening of the cavity directly into the bilayer. Moreover, the capability to produce pure and homogeneous protein has allowed previously unattainable functional characterization of STRA6 in a reconstituted system. Here, we describe detailed methods for Danio rerio STRA6 expression in insect cells, purification in detergent and reconstitution in amphipol for structural characterization by cryo-EM. Furthermore, we show reconstitution of the protein in liposomes for an in vitro proteoliposome-based assay of STRA6-mediated retinol uptake. Finally, we present methods and preliminary cryo-EM data for STRA6 incorporated in lipid-filled nanodiscs, a close to native milieu to study membrane protein structure and function.
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http://dx.doi.org/10.1016/bs.mie.2020.03.005DOI Listing
June 2021

Structural and functional characterization of the bestrophin-2 anion channel.

Nat Struct Mol Biol 2020 04 6;27(4):382-391. Epub 2020 Apr 6.

Department of Pharmacology and Physiology, University of Rochester, School of Medicine and Dentistry, Rochester, NY, USA.

The bestrophin family of calcium (Ca)-activated chloride (Cl) channels, which mediate the influx and efflux of monovalent anions in response to the levels of intracellular Ca, comprises four members in mammals (bestrophin 1-4). Here we report cryo-EM structures of bovine bestrophin-2 (bBest2) bound and unbound by Ca at 2.4- and 2.2-Å resolution, respectively. The bBest2 structure highlights four previously underappreciated pore-lining residues specifically conserved in Best2 but not in Best1, illustrating the differences between these paralogs. Structure-inspired electrophysiological analysis reveals that, although the channel is sensitive to Ca, it has substantial Ca-independent activity for Cl, reflecting the opening at the cytoplasmic restriction of the ion conducting pathway even when Ca is absent. Moreover, the ion selectivity of bBest2 is controlled by multiple residues, including those involved in gating.
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http://dx.doi.org/10.1038/s41594-020-0402-zDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7150642PMC
April 2020

Intracellular calcium leak as a therapeutic target for RYR1-related myopathies.

Acta Neuropathol 2020 06 31;139(6):1089-1104. Epub 2020 Mar 31.

Department of Physiology and Cellular Biophysics, Clyde and Helen Wu Center for Molecular Cardiology, Columbia University Irving Medical Center, New York, NY, USA.

RYR1 encodes the type 1 ryanodine receptor, an intracellular calcium release channel (RyR1) on the skeletal muscle sarcoplasmic reticulum (SR). Pathogenic RYR1 variations can destabilize RyR1 leading to calcium leak causing oxidative overload and myopathy. However, the effect of RyR1 leak has not been established in individuals with RYR1-related myopathies (RYR1-RM), a broad spectrum of rare neuromuscular disorders. We sought to determine whether RYR1-RM affected individuals exhibit pathologic, leaky RyR1 and whether variant location in the channel structure can predict pathogenicity. Skeletal muscle biopsies were obtained from 17 individuals with RYR1-RM. Mutant RyR1 from these individuals exhibited pathologic SR calcium leak and increased activity of calcium-activated proteases. The increased calcium leak and protease activity were normalized by ex-vivo treatment with S107, a RyR stabilizing Rycal molecule. Using the cryo-EM structure of RyR1 and a new dataset of > 2200 suspected RYR1-RM affected individuals we developed a method for assigning pathogenicity probabilities to RYR1 variants based on 3D co-localization of known pathogenic variants. This study provides the rationale for a clinical trial testing Rycals in RYR1-RM affected individuals and introduces a predictive tool for investigating the pathogenicity of RYR1 variants of uncertain significance.
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http://dx.doi.org/10.1007/s00401-020-02150-wDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7788518PMC
June 2020

A Novel Mouse Model of Autoimmune Thyroiditis Induced by Immunization with Adenovirus Containing Full-Length Thyroglobulin cDNA: Implications to Genetic Studies of Thyroid Autoimmunity.

Thyroid 2020 09 19;30(9):1338-1345. Epub 2020 May 19.

Division of Endocrinology, Department of Medicine, Albert Einstein College of Medicine, Bronx, New York, USA.

Thyroglobulin (TG) is a key autoantigen in autoimmune thyroid diseases (AITD). Several single nucleotide polymorphisms (SNPs) in the locus were shown to be strongly associated with disease susceptibility in both humans and mice, and autoimmune response to TG is the earliest event in the development of thyroid autoimmunity in mice. The classical model of experimental autoimmune thyroiditis (EAT) is induced by immunizing mice with TG protein together with an adjuvant to break down immune tolerance. The classical EAT model has limited utility in genetic studies of TG since it does not allow testing the effects of TG sequence variants on the development of autoimmune thyroiditis. In this study, we have immunized CBA-J mice, an EAT-susceptible strain, with an adenovirus vector encoding the full-length human TG (hTG) to generate a model of EAT in which the TG sequence can be manipulated to test AITD-associated TG SNPs. We immunized CBA-J mice with hTG-expressing adenovirus following the well-recognized experimental autoimmune Graves' disease protocol that also uses an adenovirus vector to deliver the immunogen. After hTG adenovirus immunizations, mice developed higher T cell proliferative and cytokine responses to hTG and TG2098 (a major T cell epitope in AITD) and higher titers of TG and thyroperoxidase autoantibodies compared with mice immunized with control LacZ-expressing adenovirus. The mice, however, did not develop thyroidal lymphocytic infiltration and hypothyroidism. Our data describe a novel murine model of autoimmune thyroiditis that does not require the use of adjuvants to break down tolerance and that will allow investigators to test the effects of hTG variants in the pathoetiology of Hashimoto's thyroiditis.
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http://dx.doi.org/10.1089/thy.2019.0711DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7482114PMC
September 2020

Structure of an endosomal signaling GPCR-G protein-β-arrestin megacomplex.

Nat Struct Mol Biol 2019 12 18;26(12):1123-1131. Epub 2019 Nov 18.

Department of Medicine, Duke University Medical Center, Durham, NC, USA.

Classically, G-protein-coupled receptors (GPCRs) are thought to activate G protein from the plasma membrane and are subsequently desensitized by β-arrestin (β-arr). However, some GPCRs continue to signal through G protein from internalized compartments, mediated by a GPCR-G protein-β-arr 'megaplex'. Nevertheless, the molecular architecture of the megaplex remains unknown. Here, we present its cryo-electron microscopy structure, which shows simultaneous engagement of human G protein and bovine β-arr to the core and phosphorylated tail, respectively, of a single active human chimeric β-adrenergic receptor with the C-terminal tail of the arginine vasopressin type 2 receptor (βVR). All three components adopt their canonical active conformations, suggesting that a single megaplex GPCR is capable of simultaneously activating G protein and β-arr. Our findings provide a structural basis for GPCR-mediated sustained internalized G protein signaling.
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http://dx.doi.org/10.1038/s41594-019-0330-yDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7108872PMC
December 2019

Structural basis for activity of TRIC counter-ion channels in calcium release.

Proc Natl Acad Sci U S A 2019 03 15;116(10):4238-4243. Epub 2019 Feb 15.

State Key Laboratory of Molecular Developmental Biology, Institute of Genetics and Developmental Biology, Chinese Academy of Sciences, Beijing 100101, China;

Trimeric intracellular cation (TRIC) channels are thought to provide counter-ion currents that facilitate the active release of Ca from intracellular stores. TRIC activity is controlled by voltage and Ca modulation, but underlying mechanisms have remained unknown. Here we describe high-resolution crystal structures of vertebrate TRIC-A and TRIC-B channels, both in Ca-bound and Ca-free states, and we analyze conductance properties in structure-inspired mutagenesis experiments. The TRIC channels are symmetric trimers, wherein we find a pore in each protomer that is gated by a highly conserved lysine residue. In the resting state, Ca binding at the luminal surface of TRIC-A, on its threefold axis, stabilizes lysine blockage of the pores. During active Ca release, luminal Ca depletion removes inhibition to permit the lysine-bearing and voltage-sensing helix to move in response to consequent membrane hyperpolarization. Diacylglycerol is found at interprotomer interfaces, suggesting a role in metabolic control.
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http://dx.doi.org/10.1073/pnas.1817271116DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6410872PMC
March 2019

Wavelet Denoising of High-Bandwidth Nanopore and Ion-Channel Signals.

Nano Lett 2019 02 7;19(2):1090-1097. Epub 2019 Jan 7.

Department of Physics and Astronomy , University of Pennsylvania , Philadelphia , Pennsylvania 19104 , United States.

Recent work has pushed the noise-limited bandwidths of solid-state nanopore conductance recordings to more than 5 MHz and of ion channel conductance recordings to more than 500 kHz through the use of integrated complementary metal-oxide-semiconductor (CMOS) integrated circuits. Despite the spectral spread of the pulse-like signals that characterize these recordings when a sinusoidal basis is employed, Bessel filters are commonly used to denoise these signals to acceptable signal-to-noise ratios (SNRs) at the cost of losing many of the faster temporal features. Here, we report improvements to the SNR that can be achieved using wavelet denoising instead of Bessel filtering. When combined with state-of-the-art high-bandwidth CMOS recording instrumentation, we can reduce baseline noise levels by over a factor of 4 compared to a 2.5 MHz Bessel filter while retaining transient properties in the signal comparable to this filter bandwidth. Similarly, for ion-channel recordings, we achieve a temporal response better than a 100 kHz Bessel filter with a noise level comparable to that achievable with a 25 kHz Bessel filter. Improvements in SNR can be used to achieve robust statistical analyses of these recordings, which may provide important insights into nanopore translocation dynamics and mechanisms of ion-channel function.
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http://dx.doi.org/10.1021/acs.nanolett.8b04388DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6904930PMC
February 2019

Structure-based analysis of CysZ-mediated cellular uptake of sulfate.

Elife 2018 05 24;7. Epub 2018 May 24.

Department of Physiology and Cellular Biophysics, Columbia University, New York, United States.

Sulfur, most abundantly found in the environment as sulfate (SO), is an essential element in metabolites required by all living cells, including amino acids, co-factors and vitamins. However, current understanding of the cellular delivery of SO at the molecular level is limited. CysZ has been described as a SO permease, but its sequence family is without known structural precedent. Based on crystallographic structure information, SO binding and flux experiments, we provide insight into the molecular mechanism of CysZ-mediated translocation of SO across membranes. CysZ structures from three different bacterial species display a hitherto unknown fold and have subunits organized with inverted transmembrane topology. CysZ from assembles as a trimer of antiparallel dimers and the CysZ structures from two other species recapitulate dimers from this assembly. Mutational studies highlight the functional relevance of conserved CysZ residues.
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http://dx.doi.org/10.7554/eLife.27829DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5967866PMC
May 2018

Single-channel recordings of RyR1 at microsecond resolution in CMOS-suspended membranes.

Proc Natl Acad Sci U S A 2018 02 5;115(8):E1789-E1798. Epub 2018 Feb 5.

Department of Electrical Engineering, Columbia University, New York, NY 10027;

Single-channel recordings are widely used to explore functional properties of ion channels. Typically, such recordings are performed at bandwidths of less than 10 kHz because of signal-to-noise considerations, limiting the temporal resolution available for studying fast gating dynamics to greater than 100 µs. Here we present experimental methods that directly integrate suspended lipid bilayers with high-bandwidth, low-noise transimpedance amplifiers based on complementary metal-oxide-semiconductor (CMOS) integrated circuits (IC) technology to achieve bandwidths in excess of 500 kHz and microsecond temporal resolution. We use this CMOS-integrated bilayer system to study the type 1 ryanodine receptor (RyR1), a Ca-activated intracellular Ca-release channel located on the sarcoplasmic reticulum. We are able to distinguish multiple closed states not evident with lower bandwidth recordings, suggesting the presence of an additional Ca binding site, distinct from the site responsible for activation. An extended beta distribution analysis of our high-bandwidth data can be used to infer closed state flicker events as fast as 35 ns. These events are in the range of single-file ion translocations.
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http://dx.doi.org/10.1073/pnas.1712313115DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5828579PMC
February 2018

Structural basis for catalysis at the membrane-water interface.

Biochim Biophys Acta Mol Cell Biol Lipids 2017 Nov 30;1862(11):1368-1385. Epub 2016 Nov 30.

Department of Physiology and Cellular Biophysics, Columbia University, New York, NY 10032, USA. Electronic address:

The membrane-water interface forms a uniquely heterogeneous and geometrically constrained environment for enzymatic catalysis. Integral membrane enzymes sample three environments - the uniformly hydrophobic interior of the membrane, the aqueous extramembrane region, and the fuzzy, amphipathic interfacial region formed by the tightly packed headgroups of the components of the lipid bilayer. Depending on the nature of the substrates and the location of the site of chemical modification, catalysis may occur in each of these environments. The availability of structural information for alpha-helical enzyme families from each of these classes, as well as several beta-barrel enzymes from the bacterial outer membrane, has allowed us to review here the different ways in which each enzyme fold has adapted to the nature of the substrates, products, and the unique environment of the membrane. Our focus here is on enzymes that process lipidic substrates. This article is part of a Special Issue entitled: Bacterial Lipids edited by Russell E. Bishop.
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http://dx.doi.org/10.1016/j.bbalip.2016.11.011DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5449265PMC
November 2017

Structures of the colossal RyR1 calcium release channel.

Curr Opin Struct Biol 2016 08 27;39:144-152. Epub 2016 Sep 27.

Department of Biochemistry and Molecular Biophysics, Columbia University, New York, NY 10032, USA. Electronic address:

Ryanodine receptors (RyRs) are intracellular cation channels that mediate the rapid and voluminous release of Ca from the sarcoplasmic reticulum (SR) as required for excitation-contraction coupling in cardiac and skeletal muscle. Understanding of the architecture and gating of RyRs has advanced dramatically over the past two years, due to the publication of high resolution cryo-electron microscopy (cryoEM) reconstructions and associated atomic models of multiple functional states of the skeletal muscle receptor, RyR1. Here we review recent advances in our understanding of RyR architecture and gating, and highlight remaining gaps in understanding which we anticipate will soon be filled.
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http://dx.doi.org/10.1016/j.sbi.2016.09.002DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5419430PMC
August 2016

Structural Basis for Gating and Activation of RyR1.

Cell 2016 Sep;167(1):145-157.e17

Department of Biochemistry and Molecular Biophysics, Columbia University, New York, NY 10032, USA; Howard Hughes Medical Institute, Columbia University, New York, NY 10032, USA; Department of Biological Sciences, Columbia University, New York, NY 10032, USA. Electronic address:

The type-1 ryanodine receptor (RyR1) is an intracellular calcium (Ca(2+)) release channel required for skeletal muscle contraction. Here, we present cryo-EM reconstructions of RyR1 in multiple functional states revealing the structural basis of channel gating and ligand-dependent activation. Binding sites for the channel activators Ca(2+), ATP, and caffeine were identified at interdomain interfaces of the C-terminal domain. Either ATP or Ca(2+) alone induces conformational changes in the cytoplasmic assembly ("priming"), without pore dilation. In contrast, in the presence of all three activating ligands, high-resolution reconstructions of open and closed states of RyR1 were obtained from the same sample, enabling analyses of conformational changes associated with gating. Gating involves global conformational changes in the cytosolic assembly accompanied by local changes in the transmembrane domain, which include bending of the S6 transmembrane segment and consequent pore dilation, displacement, and deformation of the S4-S5 linker and conformational changes in the pseudo-voltage-sensor domain.
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http://dx.doi.org/10.1016/j.cell.2016.08.075DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5142848PMC
September 2016

Structure of the STRA6 receptor for retinol uptake.

Science 2016 08;353(6302)

Department of Physiology and Cellular Biophysics, Columbia University, New York, NY 10032, USA.

Vitamin A homeostasis is critical to normal cellular function. Retinol-binding protein (RBP) is the sole specific carrier in the bloodstream for hydrophobic retinol, the main form in which vitamin A is transported. The integral membrane receptor STRA6 mediates cellular uptake of vitamin A by recognizing RBP-retinol to trigger release and internalization of retinol. We present the structure of zebrafish STRA6 determined to 3.9-angstrom resolution by single-particle cryo-electron microscopy. STRA6 has one intramembrane and nine transmembrane helices in an intricate dimeric assembly. Unexpectedly, calmodulin is bound tightly to STRA6 in a noncanonical arrangement. Residues involved with RBP binding map to an archlike structure that covers a deep lipophilic cleft. This cleft is open to the membrane, suggesting a possible mode for internalization of retinol through direct diffusion into the lipid bilayer.
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http://dx.doi.org/10.1126/science.aad8266DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5114850PMC
August 2016

Structures of aminoarabinose transferase ArnT suggest a molecular basis for lipid A glycosylation.

Science 2016 Feb;351(6273):608-12

Department of Physiology and Cellular Biophysics, Columbia University, New York, NY 10032, USA.

Polymyxins are antibiotics used in the last line of defense to combat multidrug-resistant infections by Gram-negative bacteria. Polymyxin resistance arises through charge modification of the bacterial outer membrane with the attachment of the cationic sugar 4-amino-4-deoxy-l-arabinose to lipid A, a reaction catalyzed by the integral membrane lipid-to-lipid glycosyltransferase 4-amino-4-deoxy-L-arabinose transferase (ArnT). Here, we report crystal structures of ArnT from Cupriavidus metallidurans, alone and in complex with the lipid carrier undecaprenyl phosphate, at 2.8 and 3.2 angstrom resolution, respectively. The structures show cavities for both lipidic substrates, which converge at the active site. A structural rearrangement occurs on undecaprenyl phosphate binding, which stabilizes the active site and likely allows lipid A binding. Functional mutagenesis experiments based on these structures suggest a mechanistic model for ArnT family enzymes.
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http://dx.doi.org/10.1126/science.aad1172DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4963604PMC
February 2016

Structure of the polyisoprenyl-phosphate glycosyltransferase GtrB and insights into the mechanism of catalysis.

Nat Commun 2016 Jan 5;7:10175. Epub 2016 Jan 5.

Department of Physiology and Cellular Biophysics, Columbia University, New York, New York 10032, USA.

The attachment of a sugar to a hydrophobic polyisoprenyl carrier is the first step for all extracellular glycosylation processes. The enzymes that perform these reactions, polyisoprenyl-glycosyltransferases (PI-GTs) include dolichol phosphate mannose synthase (DPMS), which generates the mannose donor for glycosylation in the endoplasmic reticulum. Here we report the 3.0 Å resolution crystal structure of GtrB, a glucose-specific PI-GT from Synechocystis, showing a tetramer in which each protomer contributes two helices to a membrane-spanning bundle. The active site is 15 Å from the membrane, raising the question of how water-soluble and membrane-embedded substrates are brought into apposition for catalysis. A conserved juxtamembrane domain harbours disease mutations, which compromised activity in GtrB in vitro and in human DPM1 tested in zebrafish. We hypothesize a role of this domain in shielding the polyisoprenyl-phosphate for transport to the active site. Our results reveal the basis of PI-GT function, and provide a potential molecular explanation for DPM1-related disease.
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http://dx.doi.org/10.1038/ncomms10175DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4728340PMC
January 2016

Structural basis for phosphatidylinositol-phosphate biosynthesis.

Nat Commun 2015 Oct 16;6:8505. Epub 2015 Oct 16.

Department of Physiology and Cellular Biophysics, Columbia University, New York, NY 10032, USA.

Phosphatidylinositol is critical for intracellular signalling and anchoring of carbohydrates and proteins to outer cellular membranes. The defining step in phosphatidylinositol biosynthesis is catalysed by CDP-alcohol phosphotransferases, transmembrane enzymes that use CDP-diacylglycerol as donor substrate for this reaction, and either inositol in eukaryotes or inositol phosphate in prokaryotes as the acceptor alcohol. Here we report the structures of a related enzyme, the phosphatidylinositol-phosphate synthase from Renibacterium salmoninarum, with and without bound CDP-diacylglycerol to 3.6 and 2.5 Å resolution, respectively. These structures reveal the location of the acceptor site, and the molecular determinants of substrate specificity and catalysis. Functional characterization of the 40%-identical ortholog from Mycobacterium tuberculosis, a potential target for the development of novel anti-tuberculosis drugs, supports the proposed mechanism of substrate binding and catalysis. This work therefore provides a structural and functional framework to understand the mechanism of phosphatidylinositol-phosphate biosynthesis.
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http://dx.doi.org/10.1038/ncomms9505DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4634129PMC
October 2015

Structure of a mammalian ryanodine receptor.

Nature 2015 Jan 1;517(7532):44-9. Epub 2014 Dec 1.

1] Department of Physiology and Cellular Biophysics, Columbia University, New York, New York 10032, USA [2] Department of Medicine, Columbia University, New York, New York 10032, USA [3] Wu Center for Molecular Cardiology, College of Physicians and Surgeons of Columbia University, New York, New York 10032, USA.

Ryanodine receptors (RyRs) mediate the rapid release of calcium (Ca(2+)) from intracellular stores into the cytosol, which is essential for numerous cellular functions including excitation-contraction coupling in muscle. Lack of sufficient structural detail has impeded understanding of RyR gating and regulation. Here we report the closed-state structure of the 2.3-megadalton complex of the rabbit skeletal muscle type 1 RyR (RyR1), solved by single-particle electron cryomicroscopy at an overall resolution of 4.8 Å. We fitted a polyalanine-level model to all 3,757 ordered residues in each protomer, defining the transmembrane pore in unprecedented detail and placing all cytosolic domains as tertiary folds. The cytosolic assembly is built on an extended α-solenoid scaffold connecting key regulatory domains to the pore. The RyR1 pore architecture places it in the six-transmembrane ion channel superfamily. A unique domain inserted between the second and third transmembrane helices interacts intimately with paired EF-hands originating from the α-solenoid scaffold, suggesting a mechanism for channel gating by Ca(2+).
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http://dx.doi.org/10.1038/nature13950DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4300236PMC
January 2015
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