Publications by authors named "Janesh Kumar"

32 Publications

Role of Neto1 extracellular domain in modulation of kainate receptors.

Int J Biol Macromol 2021 Oct 8. Epub 2021 Oct 8.

Laboratory of Membrane Protein Biology, National Centre for Cell Science, NCCS Complex, S. P. Pune University, Pune, Maharashtra 411007, India. Electronic address:

Kainate receptors play fundamental roles in regulating synaptic transmission and plasticity in central nervous system and are regulated by their cognate auxiliary subunits Neuropilin and tolloid-like proteins 1 and 2 (Neto). While electrophysiology-based insights into functions of Neto proteins are known, biophysical and biochemical studies into Neto proteins have been largely missing till-date. Our biochemical, biophysical, and functional characterization of the purified extracellular domain (ECD) of Neto1 protein shows that Neto1-ECD exists as monomers in solution and has a micromolar affinity for GluK2 receptors in apo state or closed state. Remarkably, the affinity was ~2.8 fold lower for receptors trapped in the desensitized state, highlighting the conformation-dependent interaction of Neto proteins with kainate receptors. SAXS analysis of the purified Neto1-ECD reveals that their dimensions are long enough to span the entire extracellular domain of kainate receptors. The shape and conformation of Neto1-ECD seems to be altered by calcium ions pointing towards its possible role in modulating Neto1 functions. Functional assays using GluK2 receptors and GluK2/GluA2 chimeric receptors reveal a differential role of Neto1 domains in modulating receptor functions. Although the desensitization rate was not affected by the Neto1-ECD, the recovery rates from the desensitized state are altered.
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http://dx.doi.org/10.1016/j.ijbiomac.2021.10.001DOI Listing
October 2021

Structural biology of ionotropic glutamate delta receptors and their crosstalk with metabotropic glutamate receptors.

Neuropharmacology 2021 09 26;196:108683. Epub 2021 Jun 26.

Laboratory of Membrane Protein Biology, National Centre for Cell Science, NCCS Complex, S. P. Pune University, Pune, Maharashtra, 411007, India. Electronic address:

Enigmatic orphan glutamate delta receptors (GluD) are one of the four classes of the ionotropic glutamate receptors (iGluRs) that play key roles in synaptic transmission and plasticity. While members of other iGluR families viz AMPA, NMDA, and kainate receptors are gated by glutamate, the GluD receptors neither bind glutamate nor evoke ligand-induced currents upon binding of glycine and D-serine. Thus, the GluD receptors were considered to function as structural proteins that facilitate the formation, maturation, and maintenance of synapses in the hippocampus and cerebellum. Recent work has revealed that GluD receptors have extensive crosstalk with metabotropic glutamate receptors (mGlus) and are also gated by their activation. The latest development of a novel optopharamcological tool and the cryoEM structures of GluD receptors would help define the molecular and chemical basis of the GluD receptor's role in synaptic physiology. This article is part of the special Issue on "Glutamate Receptors - Orphan iGluRs".
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http://dx.doi.org/10.1016/j.neuropharm.2021.108683DOI Listing
September 2021

Recent Insights into the Structure and Function of Mycobacterial Membrane Proteins Facilitated by Cryo-EM.

J Membr Biol 2021 06 5;254(3):321-341. Epub 2021 May 5.

Laboratory of Membrane Protein Biology, National Centre for Cell Science, NCCS Complex, S. P. Pune University Campus, Ganeshkhind, Pune, Maharashtra, 411007, India.

Mycobacterium tuberculosis (Mtb) is one of the deadliest pathogens encountered by humanity. Over the decades, its characteristic membrane organization and composition have been understood. However, there is still limited structural information and mechanistic understanding of the constituent membrane proteins critical for drug discovery pipelines. Recent advances in single-particle cryo-electron microscopy and cryo-electron tomography have provided the much-needed impetus towards structure determination of several vital Mtb membrane proteins whose structures were inaccessible via X-ray crystallography and NMR. Important insights into membrane composition and organization have been gained via a combination of electron tomography and biochemical and biophysical assays. In addition, till the time of writing this review, 75 new structures of various Mtb proteins have been reported via single-particle cryo-EM. The information obtained from these structures has improved our understanding of the mechanisms of action of these proteins and the physiological pathways they are associated with. These structures have opened avenues for structure-based drug design and vaccine discovery programs that might help achieve global-TB control. This review describes the structural features of selected membrane proteins (type VII secretion systems, Rv1819c, Arabinosyltransferase, Fatty Acid Synthase, F-type ATP synthase, respiratory supercomplex, ClpP1P2 protease, ClpB disaggregase and SAM riboswitch), their involvement in physiological pathways, and possible use as a drug target. Tuberculosis is a deadly disease caused by Mycobacterium tuberculosis. The Cryo-EM and tomography have simplified the understanding of the mycobacterial membrane organization. Some proteins are located in the plasma membrane; some span the entire envelope, while some, like MspA, are located in the mycomembrane. Cryo-EM has made the study of such membrane proteins feasible.
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http://dx.doi.org/10.1007/s00232-021-00179-wDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8099146PMC
June 2021

An overview of the recent advances in cryo-electron microscopy for life sciences.

Emerg Top Life Sci 2021 May;5(1):151-168

Laboratory of Membrane Protein Biology, National Centre for Cell Science, NCCS Complex, S. P. Pune University, Pune, Maharashtra 411007, India.

Cryo-electron microscopy (CryoEM) has superseded X-ray crystallography and NMR to emerge as a popular and effective tool for structure determination in recent times. It has become indispensable for the characterization of large macromolecular assemblies, membrane proteins, or samples that are limited, conformationally heterogeneous, and recalcitrant to crystallization. Besides, it is the only tool capable of elucidating high-resolution structures of macromolecules and biological assemblies in situ. A state-of-the-art electron microscope operable at cryo-temperature helps preserve high-resolution details of the biological sample. The structures can be determined, either in isolation via single-particle analysis (SPA) or helical reconstruction, electron diffraction (ED) or within the cellular environment via cryo-electron tomography (cryoET). All the three streams of SPA, ED, and cryoET (along with subtomogram averaging) have undergone significant advancements in recent times. This has resulted in breaking the boundaries with respect to both the size of the macromolecules/assemblies whose structures could be determined along with the visualization of atomic details at resolutions unprecedented for cryoEM. In addition, the collection of larger datasets combined with the ability to sort and process multiple conformational states from the same sample are providing the much-needed link between the protein structures and their functions. In overview, these developments are helping scientists decipher the molecular mechanism of critical cellular processes, solve structures of macromolecules that were challenging targets for structure determination until now, propelling forward the fields of biology and biomedicine. Here, we summarize recent advances and key contributions of the three cryo-electron microscopy streams of SPA, ED, and cryoET.
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http://dx.doi.org/10.1042/ETLS20200295DOI Listing
May 2021

CASSPER is a semantic segmentation-based particle picking algorithm for single-particle cryo-electron microscopy.

Commun Biol 2021 02 15;4(1):200. Epub 2021 Feb 15.

Artificial Intelligence Research and Intelligent Systems (airis4D), Thelliyoor, Kerala, India.

Particle identification and selection, which is a prerequisite for high-resolution structure determination of biological macromolecules via single-particle cryo-electron microscopy poses a major bottleneck for automating the steps of structure determination. Here, we present a generalized deep learning tool, CASSPER, for the automated detection and isolation of protein particles in transmission microscope images. This deep learning tool uses Semantic Segmentation and a collection of visually prepared training samples to capture the differences in the transmission intensities of protein, ice, carbon, and other impurities found in the micrograph. CASSPER is a semantic segmentation based method that does pixel-level classification and completely eliminates the need for manual particle picking. Integration of Contrast Limited Adaptive Histogram Equalization (CLAHE) in CASSPER enables high-fidelity particle detection in micrographs with variable ice thickness and contrast. A generalized CASSPER model works with high efficiency on unseen datasets and can potentially pick particles on-the-fly, enabling data processing automation.
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http://dx.doi.org/10.1038/s42003-021-01721-1DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7884729PMC
February 2021

Emerging insights into the structure and function of ionotropic glutamate delta receptors.

Br J Pharmacol 2020 Nov 4. Epub 2020 Nov 4.

Laboratory of Membrane Protein Biology, National Centre for Cell Science, NCCS Complex, S. P. Pune University, Pune, India.

Glutamate delta-1 (GluD1) and delta-2 (GluD2) receptors belong to the orphan GluD subfamily of ionotropic glutamate receptors (iGluRs). GluDs were classified as ionotropic glutamate receptors based on their sequence similarity. Two decades after these GluDs were first cloned they are still considered "orphan" due to a lack of knowledge of the endogenous ligands that can activate them. Nevertheless, they are crucial for synapse formation, maturation and maintenance of CNS functions, and are implicated in multiple neuronal disorders, including schizophrenia, autism spectrum disorder and depressive disorders. Over the last decade significant discoveries have been made, include role of GluD receptors in mediating trans-synaptic interactions and their unique non-swapped architecture, which is distinct from other ionotropic glutamate receptors. Also, the prospect of GluD ionotropic activity being regulated by direct interaction with metabotropic glutamate receptors is exciting. These discoveries will likely drive the field in the future, providing direction to GluD research.
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http://dx.doi.org/10.1111/bph.15313DOI Listing
November 2020

The architecture of GluD2 ionotropic delta glutamate receptor elucidated by cryo-EM.

J Struct Biol 2020 08 5;211(2):107546. Epub 2020 Jun 5.

Laboratory of Membrane Protein Biology, National Centre for Cell Science, NCCS Complex, S. P. Pune University, Pune, Maharashtra 411007, India. Electronic address:

GluD2 receptor belongs to the orphan delta family of glutamate receptor ion channels. These receptors play key roles in synaptogenesis and synaptic plasticity and are associated with multiple neuronal disorders like schizophrenia, autism spectrum disorder, cerebellar ataxia, intellectual disability, paraplegia, retinal dystrophy, etc. Despite the importance of these receptors in CNS, insights into full-length GluD2 receptor structure is missing till-date. Here we report cryo-electron microscopy structure of the rat GluD2 receptor in the presence of calcium ions and the ligand 7-chlorokynurenic acid, elucidating its 3D architecture. The structure reveals a non-swapped architecture at the extracellular amino-terminal (ATD), and ligand-binding domain (LBD) interface similar to that observed in GluD1; however, the organization and arrangement of the ATD and LBD domains in GluD2 are unique. While our results demonstrate that non-swapped architecture is conserved in the delta receptor family, they also highlight the differences that exist between the two member receptors; GluD1 and GluD2.
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http://dx.doi.org/10.1016/j.jsb.2020.107546DOI Listing
August 2020

Structural dynamics of the GluK3-kainate receptor neurotransmitter binding domains revealed by cryo-EM.

Int J Biol Macromol 2020 Apr 30;149:1051-1058. Epub 2020 Jan 30.

Laboratory of Membrane Protein Biology, National Centre for Cell Science, NCCS Complex, S. P. Pune University, Pune, Maharashtra 411007, India. Electronic address:

Kainate receptors belong to the ionotropic glutamate receptor family and play critical roles in the regulation of synaptic networks. The kainate receptor subunit GluK3 has unique functional properties and contributes to presynaptic facilitation at the hippocampal mossy fiber synapses along with roles at the post-synapses. To gain structural insights into the unique functional properties and dynamics of GluK3 receptor, we imaged them via electron microscopy in the apo-state and in complex with either agonist kainate or antagonist UBP301. Our analysis of all the GluK3 full-length structures not only provides insights into the receptor transitions between desensitized and closed states but also reveals a "non-classical" conformation of neurotransmitter binding domain in the closed-state distinct from that observed in AMPA and other kainate receptor structures. We show by molecular dynamics simulations that Asp759 influences the stability of the LBD dimers and hence could be responsible for the observed conformational variability and dynamics of the GluK3 via electron microscopy. Lower dimer stability could explain faster desensitization and low agonist sensitivity of GluK3. In overview, our work helps to associate biochemistry and physiology of GluK3 receptors with their structural biology and offers structural insights into the unique functional properties of these atypical receptors.
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http://dx.doi.org/10.1016/j.ijbiomac.2020.01.282DOI Listing
April 2020

Cryo-EM structures of the ionotropic glutamate receptor GluD1 reveal a non-swapped architecture.

Nat Struct Mol Biol 2020 01 10;27(1):84-91. Epub 2020 Jan 10.

Laboratory of Membrane Protein Biology, National Centre for Cell Science, NCCS Complex, S. P. Pune University, Pune, Maharashtra, India.

Ionotropic orphan delta (GluD) receptors are not gated by glutamate or any other endogenous ligand but are grouped with ionotropic glutamate receptors (iGluRs) based on sequence similarity. GluD1 receptors play critical roles in synaptogenesis and synapse maintenance and have been implicated in neuronal disorders, including schizophrenia, cognitive deficits, and cerebral ataxia. Here we report cryo-EM structures of the rat GluD1 receptor complexed with calcium and the ligand 7-chlorokynurenic acid (7-CKA), elucidating molecular architecture and principles of receptor assembly. The structures reveal a non-swapped architecture at the interface of the extracellular amino-terminal domain (ATD) and the ligand-binding domain (LBD). This finding is in contrast with structures of other families of iGluRs, where the dimer partners between the ATD and LBD layers are swapped. Our results demonstrate that principles of architecture and symmetry are not conserved between delta receptors and other iGluRs and provide a molecular blueprint for understanding the functions of the 'orphan' class of iGluRs.
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http://dx.doi.org/10.1038/s41594-019-0359-yDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7025878PMC
January 2020

Structural and Functional Insights into GluK3-kainate Receptor Desensitization and Recovery.

Sci Rep 2019 07 16;9(1):10254. Epub 2019 Jul 16.

Laboratory of Membrane Protein Biology, National Centre for Cell Science, NCCS Complex, S. P. Pune University, Maharashtra, Pune, 411007, India.

GluK3-kainate receptors are atypical members of the iGluR family that reside at both the pre- and postsynapse and play a vital role in the regulation of synaptic transmission. For a better understanding of structural changes that underlie receptor functions, GluK3 receptors were trapped in desensitized and resting/closed states and structures analyzed using single particle cryo-electron microscopy. While the desensitized GluK3 has domain organization as seen earlier for another kainate receptor-GluK2, antagonist bound GluK3 trapped a resting state with only two LBD domains in dimeric arrangement necessary for receptor activation. Using structures as a guide, we show that the N-linked glycans at the interface of GluK3 ATD and LBD likely mediate inter-domain interactions and attune receptor-gating properties. The mutational analysis also identified putative N-glycan interacting residues. Our results provide a molecular framework for understanding gating properties unique to GluK3 and exploring the role of N-linked glycosylation in their modulation.
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http://dx.doi.org/10.1038/s41598-019-46770-zDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6635489PMC
July 2019

Improved neural differentiation of normal and abnormal induced pluripotent stem cell lines in the presence of valproic acid.

J Tissue Eng Regen Med 2019 08 18;13(8):1482-1496. Epub 2019 Jul 18.

Stem Cell Laboratory, National Centre for Cell Science, Pune, India.

During the generation of induced pluripotent stem cell (iPSC) lines from cord blood CD34 cells, a line having complete trisomy of Chromosome 1 and deletion of q23 to qTer of Chromosome 11 was accidentally developed in our lab. The abnormality was consistently detected even at higher passages. These chromosomal anomalies are known to manifest neurological developmental defects. In order to examine if such defects occur during in vitro differentiation of the cell line, we set up a protocol for neural differentiation. Valproic acid (VPA) was earlier reported by us to enhance neural differentiation of placental mesenchymal stem cells. Here, we induced normal and abnormal iPSC lines to neural lineage with/without VPA. Neural differentiation was observed in all four sets, but for both the iPSCs lines, VPA sets performed better. The characteristics tested were morphology, neural filament length, detection of neural markers, and electrophysiology. In summary, the karyotypically abnormal line exhibited efficient neural differentiation. This iPSC line may serve as a useful tool to study abnormalities associated with trisomy 1 and deletion of q23 to qTer of Chromosome 11.
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http://dx.doi.org/10.1002/term.2904DOI Listing
August 2019

Seven coordinate Co(ii) and six coordinate Ni(ii) complexes of an aromatic macrocyclic triamide ligand as paraCEST agents for MRI.

Dalton Trans 2019 Jun 29;48(24):8899-8910. Epub 2019 May 29.

School of Basic Sciences, Indian Institute of Technology Bhubaneswar, Bhubaneswar, 752050, India.

We are reporting Co(ii) and Ni(ii) complexes of a pyridine containing aromatic macrocyclic triamide ligand, 3,6,9,15-tetraazabicyclo(9.3.1)pentadeca-1(15),11,13-triene-3,6,9-triacetamide (TPTA), as paramagnetic chemical exchange saturation transfer (paraCEST) MRI contrast agents. The synthesis and characterization of TPTA and its complexes are reported. The solution chemistry and solid-state structure of Co(ii) and Ni(ii) complexes are studied. Crystallographic data show that the [Co(TPTA)]·Cl·2HO complex (seven-coordinate, all four N atoms of ring and three amide O atoms) has a distorted pentagonal bipyramidal geometry, however the [Ni(TPTA)Cl]·Cl·0.25HO complex (six-coordinate, all four N atoms of the ring, one amide O and one chloride ion) adopts a distorted octahedral geometry. Notably the two pendent amide arms are not coordinated in the [Ni(TPTA)Cl] complex and one chloride ion fulfils its sixth coordination. The CEST effect of [Co(TPTA)] and [Ni(TPTA)Cl] amide protons is observed at 57 ppm and 78 ppm downfield of the bulk water proton respectively in a buffer solution containing 20 mM N-(2-hydroxyethyl)piperazine-N'-ethanesulfonic acid and 100 mM NaCl at pH 7.4 at 37 °C on a 9.4 T NMR spectrometer. The effects of CEST intensity and exchange rate constant with variation of pH of the solution were studied. The CEST effect and exchange rate constant for the amide protons of the [Co(TPTA)] complex have been monitored in HEPES buffer, fetal bovine serum (FBS), rabbit serum and 4% agarose gel (w/w). The stability of the [Co(TPTA)] complex in aqueous solution towards oxidation was verified by cyclic voltammetry measurement. The stability of [Co(TPTA)] has further been monitored in the presence of biologically relevant ions including HPO, CO, and Zn and under acidic conditions.
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http://dx.doi.org/10.1039/c9dt00747dDOI Listing
June 2019

Self-assembled monolayers improve protein distribution on holey carbon cryo-EM supports.

Sci Rep 2014 Nov 18;4:7084. Epub 2014 Nov 18.

Laboratory of Cell Biology, Center for Cancer Research, NCI, NIH, Bethesda, MD 20892.

Poor partitioning of macromolecules into the holes of holey carbon support grids frequently limits structural determination by single particle cryo-electron microscopy (cryo-EM). Here, we present a method to deposit, on gold-coated carbon grids, a self-assembled monolayer whose surface properties can be controlled by chemical modification. We demonstrate the utility of this approach to drive partitioning of ionotropic glutamate receptors into the holes, thereby enabling 3D structural analysis using cryo-EM methods.
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http://dx.doi.org/10.1038/srep07084DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4235105PMC
November 2014

Structural mechanism of glutamate receptor activation and desensitization.

Nature 2014 Oct 3;514(7522):328-34. Epub 2014 Aug 3.

Laboratory of Cell Biology, Center for Cancer Research, NCI, NIH, Bethesda, Maryland 20892, USA.

Ionotropic glutamate receptors are ligand-gated ion channels that mediate excitatory synaptic transmission in the vertebrate brain. To gain a better understanding of how structural changes gate ion flux across the membrane, we trapped rat AMPA (α-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid) and kainate receptor subtypes in their major functional states and analysed the resulting structures using cryo-electron microscopy. We show that transition to the active state involves a 'corkscrew' motion of the receptor assembly, driven by closure of the ligand-binding domain. Desensitization is accompanied by disruption of the amino-terminal domain tetramer in AMPA, but not kainate, receptors with a two-fold to four-fold symmetry transition in the ligand-binding domains in both subtypes. The 7.6 Å structure of a desensitized kainate receptor shows how these changes accommodate channel closing. These findings integrate previous physiological, biochemical and structural analyses of glutamate receptors and provide a molecular explanation for key steps in receptor gating.
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http://dx.doi.org/10.1038/nature13603DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4199900PMC
October 2014

Zinc potentiates GluK3 glutamate receptor function by stabilizing the ligand binding domain dimer interface.

Neuron 2012 Nov;76(3):565-78

University of Bordeaux, Interdisciplinary Institute for Neuroscience, UMR 5297, 33000 Bordeaux, France.

Kainate receptors (KARs) play a key role in the regulation of synaptic networks. Here, we show that zinc, a cation released at a subset of glutamatergic synapses, potentiates glutamate currents mediated by homomeric and heteromeric KARs containing GluK3 at 10-100 μM concentrations, whereas it inhibits other KAR subtypes. Potentiation of GluK3 currents is mainly due to reduced desensitization, as shown by kinetic analysis and desensitization mutants. Crystallographic and mutation analyses revealed that a specific zinc binding site is formed at the base of the ligand binding domain (LBD) dimer interface by a GluK3-specific aspartate (Asp759), together with two conserved residues, His762 and Asp730, the latter located on the partner subunit. In addition, we propose that tetrameric GluK2/GluK3 receptors are likely assembled as pairs of heterodimeric LBDs. Therefore, zinc binding stabilizes the labile GluK3 dimer interface, slows desensitization, and potentiates currents, providing a mechanism for KAR potentiation at glutamatergic synapses.
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http://dx.doi.org/10.1016/j.neuron.2012.08.027DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4132841PMC
November 2012

Functional insights from glutamate receptor ion channel structures.

Annu Rev Physiol 2013 4;75:313-37. Epub 2012 Sep 4.

Laboratory of Cellular and Molecular Neurophysiology, Porter Neuroscience Research Center, NICHD, NIH, DHHS, Bethesda, Maryland 20892, USA.

X-ray crystal structures for the soluble amino-terminal and ligand-binding domains of glutamate receptor ion channels, combined with a 3.6-Å-resolution structure of the full-length AMPA receptor GluA2 homotetramer, provide unique insights into the mechanisms of the assembly and function of glutamate receptor ion channels. Increasingly sophisticated biochemical, computational, and electrophysiological experiments are beginning to reveal the mechanism of action of partial agonists and suggest new models for the mechanism of action of allosteric modulators. Newly identified NMDA receptor ligands acting at novel sites offer hope for the development of subtype-selective modulators. The many unresolved issues include the role of the amino-terminal domain in AMPA receptor signaling and the mechanisms by which auxiliary proteins regulate receptor activity. The structural basis for ion permeation and ion channel block also remain areas of uncertainty, and despite substantial progress, molecular dynamics simulations have yet to reveal how glutamate binding opens the ion channel pore.
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http://dx.doi.org/10.1146/annurev-physiol-030212-183711DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4130219PMC
August 2013

Analysis of high-affinity assembly for AMPA receptor amino-terminal domains.

J Gen Physiol 2012 May 16;139(5):371-88. Epub 2012 Apr 16.

Laboratory of Cellular Imaging and Macromolecular Biophysics, Bioengineering and Physical Science Shared Resource, Section on Biophotonics, The National Institute of Biomedical Imaging and Bioengineering, National Institutes of Health, Department of Health and Human Services, Bethesda, MD 20892, USA.

Analytical ultracentrifugation (AUC) and steady-state fluorescence anisotropy were used to measure the equilibrium dissociation constant (Kd) for formation of dimers by the amino-terminal domains (ATDs) of the GluA2 and GluA3 subtypes of AMPA receptor. Previous reports on GluA2 dimerization differed in their estimate of the monomer-dimer Kd by a 2,400-fold range, with no consensus on whether the ATD forms tetramers in solution. We find by sedimentation velocity (SV) analysis performed using absorbance detection a narrow range of monomer-dimer Kd values for GluA2, from 5 to 11 nM for six independent experiments, with no detectable formation of tetramers and no effect of glycosylation or the polypeptide linker connecting the ATD and ligand-binding domains; for GluA3, the monomer-dimer Kd was 5.6 µM, again with no detectable tetramer formation. For sedimentation equilibrium (SE) experiments, a wide range of Kd values was obtained for GluA2, from 13 to 284 nM, whereas for GluA3, the Kd of 3.1 µM was less than twofold different from the SV value. Analysis of cell contents after the ∼1-week centrifuge run by silver-stained gels revealed low molecular weight GluA2 breakdown products. Simulated data for SE runs demonstrate that the apparent Kd for GluA2 varies with the extent of proteolysis, leading to artificially high Kd values. SV experiments with fluorescence detection for GluA2 labeled with 5,6-carboxyfluorescein, and fluorescence anisotropy measurements for GluA2 labeled with DyLight405, yielded Kd values of 5 and 11 nM, consistent with those from SV with absorbance detection. However, the sedimentation coefficients measured by AUC using absorbance and fluorescence systems were strikingly different, and for the latter are not consistent with hydrodynamic protein models. Thus, for unknown reasons, the concentration dependence of sedimentation coefficients obtained with fluorescence detection SV may be unreliable, limiting the usefulness of this technique for quantitative analysis.
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http://dx.doi.org/10.1085/jgp.201210770DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3343374PMC
May 2012

Building and breaking interfaces: how a receptor takes shape.

J Neurosci 2011 Jul;31(30):10749-51

Laboratory of Cellular and Molecular Neurophysiology, National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, Maryland 20892-3701, USA.

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http://dx.doi.org/10.1523/JNEUROSCI.2312-11.2011DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3181160PMC
July 2011

Structure and assembly mechanism for heteromeric kainate receptors.

Neuron 2011 Jul;71(2):319-31

Laboratory of Cellular and Molecular Neurophysiology, Porter Neuroscience Research Center, NICHD, NIH, DHHS, Bethesda, MD 20892, USA.

Native glutamate receptor ion channels are tetrameric assemblies containing two or more different subunits. NMDA receptors are obligate heteromers formed by coassembly of two or three divergent gene families. While some AMPA and kainate receptors can form functional homomeric ion channels, the KA1 and KA2 subunits are obligate heteromers which function only in combination with GluR5-7. The mechanisms controlling glutamate receptor assembly involve an initial step in which the amino terminal domains (ATD) assemble as dimers. Here, we establish by sedimentation velocity that the ATDs of GluR6 and KA2 coassemble as a heterodimer of K(d) 11 nM, 32,000-fold lower than the K(d) for homodimer formation by KA2; we solve crystal structures for the GluR6/KA2 ATD heterodimer and heterotetramer assemblies. Using these structures as a guide, we perform a mutant cycle analysis to probe the energetics of assembly and show that high-affinity ATD interactions are required for biosynthesis of functional heteromeric receptors.
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http://dx.doi.org/10.1016/j.neuron.2011.05.038DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3145919PMC
July 2011

A highly conserved protein of unknown function in Sinorhizobium meliloti affects sRNA regulation similar to Hfq.

Nucleic Acids Res 2011 Jun 15;39(11):4691-708. Epub 2011 Feb 15.

Department of Biology, Massachusetts Institute of Technology, Cambridge, MA 02139-4307, USA.

The SMc01113/YbeY protein, belonging to the UPF0054 family, is highly conserved in nearly every bacterium. However, the function of these proteins still remains elusive. Our results show that SMc01113/YbeY proteins share structural similarities with the MID domain of the Argonaute (AGO) proteins, and might similarly bind to a small-RNA (sRNA) seed, making a special interaction with the phosphate on the 5'-side of the seed, suggesting they may form a component of the bacterial sRNA pathway. Indeed, eliminating SMc01113/YbeY expression in Sinorhizobium meliloti produces symbiotic and physiological phenotypes strikingly similar to those of the hfq mutant. Hfq, an RNA chaperone, is central to bacterial sRNA-pathway. We evaluated the expression of 13 target genes in the smc01113 and hfq mutants. Further, we predicted the sRNAs that may potentially target these genes, and evaluated the accumulation of nine sRNAs in WT and smc01113 and hfq mutants. Similar to hfq, smc01113 regulates the accumulation of sRNAs as well as the target mRNAs. AGOs are central components of the eukaryotic sRNA machinery and conceptual parallels between the prokaryotic and eukaryotic sRNA pathways have long been drawn. Our study provides the first line of evidence for such conceptual parallels. Furthermore, our investigation gives insights into the sRNA-mediated regulation of stress adaptation in S. meliloti.
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http://dx.doi.org/10.1093/nar/gkr060DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3113577PMC
June 2011

Crystal structures of the glutamate receptor ion channel GluK3 and GluK5 amino-terminal domains.

J Mol Biol 2010 Dec 14;404(4):680-96. Epub 2010 Oct 14.

Laboratory of Cellular and Molecular Neurophysiology, Porter Neuroscience Research Center, National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, MD 20892, USA.

Ionotropic glutamate receptors (iGluRs) mediate the majority of fast excitatory synaptic neurotransmission in the central nervous system. The selective assembly of iGluRs into AMPA, kainate, and N-methyl-d-aspartic acid (NMDA) receptor subtypes is regulated by their extracellular amino-terminal domains (ATDs). Kainate receptors are further classified into low-affinity receptor families (GluK1-GluK3) and high-affinity receptor families (GluK4-GluK5) based on their affinity for the neurotoxin kainic acid. These two families share a 42% sequence identity for the intact receptor but only a 27% sequence identity at the level of ATD. We have determined for the first time the high-resolution crystal structures of GluK3 and GluK5 ATDs, both of which crystallize as dimers but with a strikingly different dimer assembly at the R1 interface. By contrast, for both GluK3 and GluK5, the R2 domain dimer assembly is similar to those reported previously for other non-NMDA iGluRs. This observation is consistent with the reports that GluK4-GluK5 cannot form functional homomeric ion channels and require obligate coassembly with GluK1-GluK3. Our analysis also reveals that the relative orientation of domains R1 and R2 in individual non-NMDA receptor ATDs varies by up to 10°, in contrast to the 50° difference reported for the NMDA receptor GluN2B subunit. This restricted domain movement in non-NMDA receptor ATDs seems to result both from extensive intramolecular contacts between domain R1 and domain R2 and from their assembly as dimers, which interact at both R1 and R2 domains. Our results provide the first insights into the structure and function of GluK4-GluK5, the least understood family of iGluRs.
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http://dx.doi.org/10.1016/j.jmb.2010.10.006DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2991425PMC
December 2010

Domain organization and function in GluK2 subtype kainate receptors.

Proc Natl Acad Sci U S A 2010 May 19;107(18):8463-8. Epub 2010 Apr 19.

Laboratory of Cellular and Molecular Neurophysiology, Department of Health and Human Services, Porter Neuroscience Research Center, National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, MD 20892, USA.

Glutamate receptor ion channels (iGluRs) are excitatory neurotransmitter receptors with a unique molecular architecture in which the extracellular domains assemble as a dimer of dimers. The structure of individual dimer assemblies has been established previously for both the isolated ligand-binding domain (LBD) and more recently for the larger amino terminal domain (ATD). How these dimers pack to form tetrameric assemblies in intact iGluRs has remained controversial. Using recently solved crystal structures for the GluK2 kainate receptor ATD as a guide, we performed cysteine mutant cross-linking experiments in full-length tetrameric GluK2 to establish how the ATD packs in a dimer of dimers assembly. A similar approach, using a full-length AMPA receptor GluA2 crystal structure as a guide, was used to design cysteine mutant cross-links for the GluK2 LBD dimer of dimers assembly. The formation of cross-linked tetramers in full-length GluK2 by combinations of ATD and LBD mutants which individually produce only cross-linked dimers suggests that subunits in the ATD and LBD layers swap dimer partners. Functional studies reveal that cross-linking either the ATD or the LBD inhibits activation of GluK2 and that, in the LBD, cross-links within and between dimers have different effects. These results establish that kainate and AMPA receptors have a conserved extracellular architecture and provide insight into the role of individual dimer assemblies in activation of ion channel gating.
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http://dx.doi.org/10.1073/pnas.1000838107DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2889583PMC
May 2010

The N-terminal domain of GluR6-subtype glutamate receptor ion channels.

Nat Struct Mol Biol 2009 Jun 24;16(6):631-8. Epub 2009 May 24.

Laboratory of Cellular and Molecular Neurophysiology, Porter Neuroscience Research Center, National Institute of Child Health and Human Development, Bethesda, Maryland, USA.

The amino-terminal domain (ATD) of glutamate receptor ion channels, which controls their selective assembly into AMPA, kainate and NMDA receptor subtypes, is also the site of action of NMDA receptor allosteric modulators. Here we report the crystal structure of the ATD from the kainate receptor GluR6. The ATD forms dimers in solution at micromolar protein concentrations and crystallizes as a dimer. Unexpectedly, each subunit adopts an intermediate extent of domain closure compared to the apo and ligand-bound complexes of LIVBP and G protein-coupled glutamate receptors (mGluRs), and the dimer assembly has a markedly different conformation from that found in mGluRs. This conformation is stabilized by contacts between large hydrophobic patches in the R2 domain that are absent in NMDA receptors, suggesting that the ATDs of individual glutamate receptor ion channels have evolved into functionally distinct families.
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http://dx.doi.org/10.1038/nsmb.1613DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2729365PMC
June 2009

Structure of the buffalo secretory signalling glycoprotein at 2.8 A resolution.

Acta Crystallogr Sect F Struct Biol Cryst Commun 2007 Apr 12;63(Pt 4):258-65. Epub 2007 Mar 12.

Department of Biophysics, All India Institute of Medical Sciences, New Delhi 110029, India.

The crystal structure of a 40 kDa signalling glycoprotein from buffalo (SPB-40) has been determined at 2.8 A resolution. SPB-40 acts as a protective signalling factor by binding to viable cells during the early phase of involution, during which extensive tissue remodelling occurs. It was isolated from the dry secretions of Murrah buffalo. It was purified and crystallized using the hanging-drop vapour-diffusion method with 19% ethanol as the precipitant. The protein was also cloned and its complete nucleotide and amino-acid sequences were determined. When compared with the sequences of other members of the family, the sequence of SPB-40 revealed two very important mutations in the sugar-binding region, in which Tyr120 changed to Trp120 and Glu269 changed to Trp269. The structure showed a significant distortion in the shape of the sugar-binding groove. The water structure in the groove is also drastically altered. The folding of the protein chain in the flexible region comprising segments His188-His197, Phe202-Arg212 and Tyr244-Pro260 shows large variations when compared with other proteins of the family.
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http://dx.doi.org/10.1107/S1744309107010445DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2330205PMC
April 2007

Carbohydrate-binding properties of goat secretory glycoprotein (SPG-40) and its functional implications: structures of the native glycoprotein and its four complexes with chitin-like oligosaccharides.

Acta Crystallogr D Biol Crystallogr 2007 Apr 16;63(Pt 4):437-46. Epub 2007 Mar 16.

Department of Biophysics, All India Institute of Medical Sciences, New Delhi 110029, India.

A 40 kDa glycoprotein (SPG-40) secreted during involution works as a protective signalling factor through its binding to viable cells. The crystal structure of the native protein has been determined at 2.3 A resolution. This is the first report on the carbohydrate-binding properties of SPG-40; the structure determinations of the complexes of SPG-40 with four oligosaccharides of different lengths at resolutions ranging from 2.2 to 2.8 A are described. Carbohydrate-binding studies with N-acetylglucosamines (GlcNAc(n), n = 3-6) using fluorescence spectroscopy revealed poor binding effects with GlcNAc(3) and GlcNAc(4), while GlcNAc(5) and GlcNAc(6) bound to SPG-40 with considerable strength; the dissociation constants (K(d)) were estimated to be 260 +/- 3 and 18 +/- 4 microM, respectively. SPG-40 was cocrystallized with GlcNAc(3), GlcNAc(4), GlcNAc(5) and GlcNAc(6). The overall structure of native SPG-40 was essentially similar to that reported previously at low resolution. The structures of its complexes with GlcNAc(3), GlcNAc(4), GlcNAc(5) and GlcNAc(6) revealed the positions of these oligosaccharides in the carbohydrate-binding groove and provided insights into the mechanism of binding of oligosaccharides to SPG-40, indicating that the preferred subsites in the carbohydrate-binding groove of SPG-40 were from -4 to -2. The structure of the protein remained unperturbed upon binding of GlcNAc(3) and GlcNAc(4), but the structure changed significantly upon binding of GlcNAc(5) and GlcNAc(6). Significant conformational variations were observed in the sugar-binding groove: Trp78 partially flipped out of the barrel in GlcNAc(5), while in the GlcNAc(6) complex a completely flipped-out Trp78 was observed along with several other conformational changes, including those of Asp186 and Arg242. Such changes upon binding to carbohydrates have not previously been observed in chitin-hydrolyzing chitinases and reflect less favourable binding of carbohydrates to SPG-40. As this appears to essentially be a binding protein, this loss of binding affinity might be compensated by other intermolecular interactions such as protein-protein interactions and also by the binding of its own glycan chain.
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http://dx.doi.org/10.1107/S0907444907001631DOI Listing
April 2007

Carbohydrate binding properties and carbohydrate induced conformational switch in sheep secretory glycoprotein (SPS-40): crystal structures of four complexes of SPS-40 with chitin-like oligosaccharides.

J Struct Biol 2007 Jun 17;158(3):255-66. Epub 2006 Nov 17.

Department of Biophysics, All India Institute of Medical Sciences, New Delhi 110029, India.

Crystal structures of four complexes of sheep secretory glycoprotein (SPS-40) with N-acetylglucosamine oligosaccharides (GlcNAc(n), (n=3-6)) have been determined at moderate resolutions. The binding studies of SPS-40 have been carried out using fluorescence spectroscopy and Surface Plasmon Resonance (SPR). Structure determinations of four complexes have shown a novel binding pattern of GlcNAc(n) molecules to SPS-40. The results indicate that the most preferred recognition region in the carbohydrate binding groove in SPS-40 is at subsites -4 to -2 among which subsite -2 provides the maximum interactions with carbohydrate residues. These structures have also shown that the interactions of GlcNAc3 and GlcNAc4 do not perturb the protein structure and those of GlcNAc5 induce partial conformational changes while in the case of GlcNAc6 the partially closed binding groove opened up completely. As in other SPX-40 structures, SPS-40 structure contains three overlapping flexible surface segments, His188-His197, Phe202-Arg212 and Phe244-Pro260 with several charged residues protruding outwardly. It creates a cluster of positive charges with a flexible base thus indicating a good scope of promoting the intermolecular interactions. This protein is glycosylated at Asn39 and may recognize other receptors having sugar binding sites. It appears that SPS-40 may involve both carbohydrate and protein bindings. The systematic carbohydrate-binding studies and the detailed structural results of four protein-carbohydrate complexes provide an excellent insight into the mechanism of carbohydrate binding. These are the first studies of this kind on secretory glycoproteins and their interactions with carbohydrates.
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http://dx.doi.org/10.1016/j.jsb.2006.11.002DOI Listing
June 2007

Structure of a bovine secretory signalling glycoprotein (SPC-40) at 2.1 Angstrom resolution.

Acta Crystallogr D Biol Crystallogr 2006 Sep 19;62(Pt 9):953-63. Epub 2006 Aug 19.

Department of Biophysics, All India Institute of Medical Sciences, New Delhi 110029, India.

A recently discovered new class of 40 kDa glycoproteins forms a major component of the secretory proteins in the dry secretions of non-lactating animals. These proteins are implicated as protective signalling factors that determine which cells are to survive during the processes of drastic tissue remodelling. In order to understand its role in the remodelling of mammary glands, the detailed three-dimensional structure of the bovine signalling glycoprotein (SPC-40) has been determined using X-ray crystallography. SPC-40 was purified from bovine dry secretions and crystallized using the hanging-drop vapour-diffusion method. The crystals belong to the orthorhombic space group P2(1)2(1)2(1), with unit-cell parameters a = 62.6, b = 67.4, c = 106.9 Angstrom. The protein was also cloned in order to determine its complete amino-acid sequence. Its three-dimensional structure has been determined using data to 2.1 Angstrom resolution. The amino-acid sequence determination of SPC-40 reveals two potential N-glycosylation sites at Asn39 and Asn345, but electron density for a glycan chain was only present at Asn39. The protein adopts a conformation with the classical (beta/alpha)(8)-barrel fold of triosephosphate isomerase (TIM barrel; residues 1-237 and 310-360) with the insertion of a small alpha+beta domain (residues 240-307) similar to that observed in chitinases. However, the substitution of Leu for Glu in the consensus catalytic sequence in SPC-40 caused a loss of chitinase activity. Furthermore, the chitin-binding groove in SPC-40 is considerably distorted owing to unfavourable conformations of several residues, including Trp78, Tyr120, Asp186 and Arg242. Three surface loops, His188-His197, Phe202-Arg212 and Tyr244-Pro260, have exceptionally high B factors, suggesting large-scale flexibility. Fluorescence studies indicate that various sugars bind to SPC-40 with low affinities.
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http://dx.doi.org/10.1107/S0907444906020427DOI Listing
September 2006

Crystal structure of a secretory signalling glycoprotein from sheep at 2.0A resolution.

J Struct Biol 2006 Dec 8;156(3):505-16. Epub 2006 Jun 8.

Department of Biophysics, All India Institute of Medical Sciences, Ansari Nagar, New Delhi 110029, India.

A 40kDa glycoprotein from dry secretion of sheep is implicated as a signaling factor and is named as SPS-40. This protein is secreted only during the early phase of involution when the drastic tissue remodeling occurs in the mammary gland. SPS-40 was purified from sheep dry secretions and crystallized using hanging drop vapour diffusion method. The crystals belong to orthorhombic space group P2(1)2(1)2(1) with cell dimensions, a=62.7A, b=66.4A, c=107.5A. The protein was also cloned for the determination of its complete amino acid sequence. The three-dimensional structure of SPS-40 was determined by X-ray crystallographic method at 2.0A resolution. The structure revealed the presence of an N-linked glycan chain at Asn39. The protein adopts a conformation with a classical (beta/alpha)(8)-barrel fold of triosephosphate isomerase (TIM) (residues 1-237 and 310-360) with an insertion of a small (alpha+beta) domain (residues 240-307) similar to that observed in chitinases. However, the Leu substitution for Glu in the consensus catalytic sequence in SPS-40 causes a loss of chitinase activity. Furthermore, the sugar-binding groove in SPS-40 is distorted considerably from the standard chitin-binding site in chitinase enzymes and hence the binding of chitin-like oligosaccharides is considerably hampered. Three surface loops, His188-His197, Phe202-Arg212 and Phe244-Pro260 have exceptionally high values of B-factors (average=70.5A(2)), indicating the presence of a less defined region.
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http://dx.doi.org/10.1016/j.jsb.2006.05.008DOI Listing
December 2006

Crystal structure of schistatin, a disintegrin homodimer from saw-scaled viper (Echis carinatus) at 2.5 A resolution.

J Mol Biol 2004 Aug;341(3):829-37

Department of Biophysics, All India Institute of Medical Services, Ansari Nagar, New Dehli, India.

This is the first structure of a biological homodimer of disintegrin. Disintegrins are a class of small (4-14 kDa) proteins that bind to transmembrane integrins selectively. The present molecule is the first homodimer that has been isolated from the venom of Echis carinatus. The monomeric chain contains 64 amino acid residues. The three-dimensional structure of schistatin has been determined by the multiple isomorphous replacement method. It has been refined to an R-factor of 0.190 using all the data to 2.5 A resolution. The two subunits of the disintegrin homodimer are related by a 2-fold crystallographic symmetry. Thus, the crystallographic asymmetric unit contains a monomer of disintegrin. The monomer folds into an up-down topology with three sets of antiparallel beta-strands. The structure is well ordered with four intramolecular disulfide bonds. the two monomers are firmly linked to each other through two intermolecular disulfide bridges at their N termini together with several other interactions. This structure has corrected the error in the disulfide bond pattern of the two intermolecular disulfide bridges that was reported earlier using chemical methods. Unique sequence and structural features of the schistatin monomers suggest that they have the ability to bind well with both alphaIIb beta3 and alphav beta3 integrins. The N termini anchored two chains of the dimer diverge away at their C termini exposing the Arg-Gly-Asp motif into opposite directions thus enhancing their binding efficiency to integrins. This is one of the unique features of the present disintegrin homodimer and seems to be responsible for the clustering of integrin molecules. The homodimer binds to integrins apparently with a higher affinity than the monomers and also plays a role in the signaling pathway.
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http://dx.doi.org/10.1016/j.jmb.2004.06.048DOI Listing
August 2004
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