The Experts below are selected from a list of 52602 Experts worldwide ranked by ideXlab platform
Vasanthi Jayaraman - One of the best experts on this subject based on the ideXlab platform.
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stargazin promotes closure of the ampa receptor Ligand Binding Domain
The Journal of General Physiology, 2014Co-Authors: David M Maclean, Swarna S Ramaswamy, James R Howe, Vasanthi JayaramanAbstract:Transmembrane AMPA receptor (AMPAR) regulatory proteins (TARPs) markedly enhance AMPAR function, altering Ligand efficacy and receptor gating kinetics and thereby shaping the postsynaptic response. The structural mechanism underlying TARP effects on gating, however, is unknown. Here we find that the prototypical member of the TARP family, stargazin or γ-2, rescues gating deficits in AMPARs carrying mutations that destabilize the closed-cleft states of the Ligand-Binding Domain (LBD), suggesting that stargazin reverses the effects of these mutations and likely stabilizes closed LBD states. Furthermore, stargazin promotes a more closed conformation of the LBD, as indicated by reduced accessibility to the large antagonist NBQX. Consistent with the functional studies, luminescence resonance energy transfer experiments directly demonstrate that the AMPAR LBD is on average more closed in the presence of stargazin, in both the apo and agonist-bound states. The additional cleft closure and/or stabilization of the more closed-cleft states of the LBD is expected to translate to higher agonist efficacy and could contribute to the structural mechanism for stargazin modulation of AMPAR function.
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A LRET Based Method To Studying Intersubunit Conformational Changes In The Ligand Binding Domain Of A Functional AMPA Receptor
Biophysical Journal, 2009Co-Authors: Jennifer M. Reingle Gonzalez, Vasanthi JayaramanAbstract:Ionotropic glutamate receptors are the main excitatory neurotransmitter receptors in the mammalian central nervous system. During activation of the receptor, an agonist binds to an extracellular Domain initiating a sequence of conformational changes leading to the opening of a cation-selective channel, which subsequently closes during desensitization. Structures of the isolated Ligand Binding Domain of the AMPA subtype of the receptor have provided the first clues of the structural movements within the Ligand Binding Domain; however, these structures lack the crucial functional portion of the protein, the transmembrane segments. Additionally, these limited structures do not reveal the structural changes associated with desensitization, unless artificially decoupled with a disulfide bond. In order to determine how the agonist controls receptor activation and desensitization, it is necessary to investigate the changes in the Ligand Binding Domain in the presence of the transmembrane segments. We have modified a functional AMPA receptor (ΔN∗-AMPA) to serve as an LRET based probe that allows us to measure distance changes of the Ligand Binding Domain in the presence of the transmembrane segments. This receptor has been modified such that fluorophores can be introduced at defined sites to serve as a readout of intersubunit distance measurements associated with the apo, activated, and desensitized state. These investigations suggest that the apo state in the presence of the transmembrane segments is decoupled, and during activation, the interface is coupled due to the driving force of cleft closure, thereby stabilizing the open channel, and then the interface decouples thus leading to desensitization.
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LRET Investigations of Conformational Changes in the Ligand Binding Domain of a Functional AMPA Receptor
Biochemistry, 2008Co-Authors: Jennifer M. Reingle Gonzalez, Anu Rambhadran, Vasanthi JayaramanAbstract:The structural investigations using the soluble Ligand Binding Domain of the AMPA subtype of the glutamate receptor have provided invaluable insight into the mechanistic pathway by which agonist Binding to this extracellular Domain mediates the formation of cation-selective channels in this protein. These structures, however, are in the absence of the transmembrane segments, the primary functional component of the protein. Here, we have used a modified luminescence resonance energy transfer based method to obtain distance changes due to agonist Binding in the Ligand Binding Domain in the presence of the transmembrane segments. These distance changes show that the cleft closure conformational change observed in the isolated Ligand Binding Domain upon Binding agonist is conserved in the receptor with the channel segments, thus establishing that the isolated Ligand Binding Domain is a good model of the Domain in the receptor containing the transmembrane segments.
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Luminescence Resonance Energy Transfer Investigation of Conformational Changes in the Ligand Binding Domain of a Kainate Receptor
The Journal of biological chemistry, 2008Co-Authors: Anu Rambhadran, Vasanthi JayaramanAbstract:The apo state structure of the isolated Ligand Binding Domain of the GluR6 subunit and the conformational changes induced by agonist Binding to this protein have been investigated by luminescence resonance energy transfer (LRET) measurements. The LRET-based distances show that agonist Binding induces cleft closure, and the extent of cleft closure is proportional to the extent of activation over a wide range of activations, thus establishing that the cleft closure conformational change is one of the mechanisms by which the agonist mediates receptor activation. The LRET distances also provide insight into the apo state structure, for which there is currently no crystal structure available. The distance change between the glutamate-bound state and the apo state is similar to that observed between the glutamate-bound and antagonist UBP-310-bound form of the GluR5 Ligand Binding Domain, indicating that the cleft for the apo state of the GluR6 Ligand Binding Domain should be similar to the UBP-310-bound form of GluR5. This observation implies that te apo state of GluR6 undergoes a cleft closure of 29–30° upon Binding full agonists, one of the largest observed in the glutamate receptor family.
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Luminescence Resonance Energy Transfer Investigation of Conformational Changes in the Ligand Binding Domain of a
2008Co-Authors: Anu Rambhadran, Vasanthi JayaramanAbstract:The apo state structure of the isolated Ligand Binding Domain of the GluR6 subunit and the conformational changes induced by agonist Binding to this protein have been investigated by luminescence resonance energy transfer (LRET) measurements. The LRET-based distances show that agonist Binding induces cleft closure, and the extent of cleft closure is proportional to the extent of activation over a wide range of activations, thus establishing that the cleft closure conformational change is one of the mechanisms by which the agonist mediates receptor activation. The LRET distances also provide insight into the apo state structure, for which there is currently no crystal structure available. The distance change between the glutamate-bound state and the apo state is similar to that observed between the glutamate-bound and antagonist UBP-310-bound form of the GluR5 Ligand Binding Domain, indicating that the cleft for the apo state of the GluR6 Ligand Binding Domain should be similar to the UBP-310bound form of GluR5. This observation implies that the apo state of GluR6 undergoes a cleft closure of 29 –30° upon Binding full agonists, one of the largest observed in the glutamate receptor family.
Dino Moras - One of the best experts on this subject based on the ideXlab platform.
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Structural and Functional Insights into the Ligand-Binding Domain of a Nonduplicated Retinoid X Nuclear Receptor from the Invertebrate Chordate Amphioxus.
Journal of Biological Chemistry, 2009Co-Authors: Giuseppe Tocchini-valentini, Dino Moras, Natacha Rochel, Hector Escriva, Pierre Germain, Carole Peluso-iltis, Mathilde Paris, Sarah Sanglier-cianferani, Alain Van Dorsselaer, Vincent LaudetAbstract:Retinoid X nuclear receptors (RXRs), as well as their insect orthologue, ultraspiracle protein (USP), play an important role in the transcription regulation mediated by the nuclear receptors as the common partner of many other nuclear receptors. Phylogenetic and structural studies have shown that the several evolutionary shifts have modified the Ligand Binding ability of RXRs. To understand the vertebrate-specific character of RXRs, we have studied the RXR Ligand-Binding Domain of the cephalochordate amphioxus (Branchiostoma floridae), an invertebrate chordate that predates the genome duplication that produced the three vertebrates RXRs (alpha, beta, and gamma). Here we report the crystal structure of a novel apotetramer conformation of the AmphiRXR Ligand-Binding Domain, which shows some similarity with the structures of the arthropods RXR/USPs. AmphiRXR adopts an apo antagonist conformation with a peculiar conformation of helix H11 filling the Binding pocket. In contrast to the arthropods RXR/USPs, which cannot be activated by any RXR Ligands, our functional data show that AmphiRXR, like the vertebrates/mollusk RXRs, is able to bind and be activated by RXR Ligands but less efficiently than vertebrate RXRs. Our data suggest that amphioxus RXR is, functionally, an intermediate between arthropods RXR/USPs and vertebrate RXRs.
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Functional and structural characterization of the insertion region in the Ligand Binding Domain of the vitamin D nuclear receptor.
European journal of biochemistry, 2001Co-Authors: Natachu Rochel, Kari Juntunen, Pirkko Vihko, G. Tocchini-valentini, Pascal F. Egea, Jean-marie Garnier, Dino MorasAbstract:Vitamin D nuclear receptor mediates the genomic actions of the active form of vitamin D, 1,25(OH)2D3. This hormone is involved in calcium and phosphate metabolism and cell differentiation. Compared to other nuclear receptors, VDR presents a large insertion region at the N-terminal part of the Ligand Binding Domain between helices H1 and H3, encoded by an additional exon. This region is poorly conserved in VDR in different species and is not well ordered as observed by secondary structure prediction. We engineered a VDR Ligand Binding Domain mutant by removing this insertion region. Here we report its biochemical and biophysical characterization. The mutant protein exhibits the same Ligand Binding, dimerization with retinoid X receptor and transactivation properties as the wild-type VDR, suggesting that the insertion region does not affect these main functions. Solution studies by small angle X-ray scattering shows that the conformation in solution of the VDR mutant is similar to that observed in the crystal and that the insertion region in the VDR wild-type is not well ordered.
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Large-scale expression and purification of the human vitamin D receptor and its Ligand-Binding Domain for structural studies.
Biochemical Journal, 1999Co-Authors: Kari Juntunen, Dino Moras, Natacha Rochel, Pirkko VihkoAbstract:We have expressed recombinant human vitamin D receptor and its Ligand-Binding Domain in Spodoptera frugiperda (Sf9) insect cells with a 30-litre bioreactor. Both proteins were purified to apparent homogeneity with yields of 0.5-3.5 mg/l. Gel-filtration analyses indicated that the purified human vitamin D receptor and its Ligand-Binding Domain were present as monomers in solution. The purified vitamin D receptor and its Ligand-Binding Domain were demonstrated to bind 1alpha,25-dihydroxyvitamin D(3) with high affinity, the K(d) values ranging from 0.9 to 1.2 nM. Neutron scattering studies of the Ligand-Binding Domain demonstrated that the samples are homogeneous and contain monomeric species of polypeptides. The purified vitamin D receptor binds to the vitamin D response elements of osteopontin and osteocalcin genes as a homodimer or as a heterodimer with the retinoid X receptor-alphaDeltaAB and we were able to purify these complexes in quantities sufficient for crystallization studies. The results indicate that we can produce biologically active human vitamin D receptor and its Ligand-Binding Domain in insect cells and purify them for functional and structural studies.
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the nuclear receptor Ligand Binding Domain structure and function
Current Opinion in Cell Biology, 1998Co-Authors: Dino Moras, Hinrich GronemeyerAbstract:Abstract In the past few years our understanding of nuclear receptor action has dramatically improved as a result of the elucidation of the crystal structures of the empty (apo) Ligand-Binding Domains of the nuclear receptor and of complexes formed by the nuclear receptor's Ligand-Binding Domain bound to agonists and antagonists. Furthermore, the concomitant identification and functional analysis of co-regulators (transcriptional intermediary factors [TIFs], comprising co-activators and co-repressors) previously predicted from squelching studies, have deepened this understanding. Recent data have provided the structural basis for the specific recognition of Ligands and the molecular mechanisms of agonism and antagonism, enabling us to gain a comprehensive view of the early steps of nuclear receptor action.
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crystal structure of the rar gamma Ligand Binding Domain bound to all trans retinoic acid
Nature, 1995Co-Authors: Jeanpaul Renaud, Marc Ruff, Pierre Chambon, Hinrich Gronemeyer, Natacha Rochel, Valeria Vivat, Dino MorasAbstract:The 2.0-A crystal structure of the Ligand-Binding Domain (LBD) of the human retinoic acid receptor (RAR)-γ bound to all-trans retinoic acid reveals the Ligand-Binding interactions and suggests an electrostatic guidance mechanism. The overall fold is similar to that of the human RXR-α apo-LBD, except for the carboxy-terminal part which folds back towards the LBD core, contributing to the hydrophobic Ligand pocket and 'sealing' its entry site. We propose a 'mouse trap' mechanism whereby a Ligand-induced conformational transition repositions the amphi-pathic α-helix of the AF-2 activating Domain and forms a transcriptionally active receptor.
Natacha Rochel - One of the best experts on this subject based on the ideXlab platform.
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Structural and Functional Insights into the Ligand-Binding Domain of a Nonduplicated Retinoid X Nuclear Receptor from the Invertebrate Chordate Amphioxus.
Journal of Biological Chemistry, 2009Co-Authors: Giuseppe Tocchini-valentini, Dino Moras, Natacha Rochel, Hector Escriva, Pierre Germain, Carole Peluso-iltis, Mathilde Paris, Sarah Sanglier-cianferani, Alain Van Dorsselaer, Vincent LaudetAbstract:Retinoid X nuclear receptors (RXRs), as well as their insect orthologue, ultraspiracle protein (USP), play an important role in the transcription regulation mediated by the nuclear receptors as the common partner of many other nuclear receptors. Phylogenetic and structural studies have shown that the several evolutionary shifts have modified the Ligand Binding ability of RXRs. To understand the vertebrate-specific character of RXRs, we have studied the RXR Ligand-Binding Domain of the cephalochordate amphioxus (Branchiostoma floridae), an invertebrate chordate that predates the genome duplication that produced the three vertebrates RXRs (alpha, beta, and gamma). Here we report the crystal structure of a novel apotetramer conformation of the AmphiRXR Ligand-Binding Domain, which shows some similarity with the structures of the arthropods RXR/USPs. AmphiRXR adopts an apo antagonist conformation with a peculiar conformation of helix H11 filling the Binding pocket. In contrast to the arthropods RXR/USPs, which cannot be activated by any RXR Ligands, our functional data show that AmphiRXR, like the vertebrates/mollusk RXRs, is able to bind and be activated by RXR Ligands but less efficiently than vertebrate RXRs. Our data suggest that amphioxus RXR is, functionally, an intermediate between arthropods RXR/USPs and vertebrate RXRs.
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Large-scale expression and purification of the human vitamin D receptor and its Ligand-Binding Domain for structural studies.
Biochemical Journal, 1999Co-Authors: Kari Juntunen, Dino Moras, Natacha Rochel, Pirkko VihkoAbstract:We have expressed recombinant human vitamin D receptor and its Ligand-Binding Domain in Spodoptera frugiperda (Sf9) insect cells with a 30-litre bioreactor. Both proteins were purified to apparent homogeneity with yields of 0.5-3.5 mg/l. Gel-filtration analyses indicated that the purified human vitamin D receptor and its Ligand-Binding Domain were present as monomers in solution. The purified vitamin D receptor and its Ligand-Binding Domain were demonstrated to bind 1alpha,25-dihydroxyvitamin D(3) with high affinity, the K(d) values ranging from 0.9 to 1.2 nM. Neutron scattering studies of the Ligand-Binding Domain demonstrated that the samples are homogeneous and contain monomeric species of polypeptides. The purified vitamin D receptor binds to the vitamin D response elements of osteopontin and osteocalcin genes as a homodimer or as a heterodimer with the retinoid X receptor-alphaDeltaAB and we were able to purify these complexes in quantities sufficient for crystallization studies. The results indicate that we can produce biologically active human vitamin D receptor and its Ligand-Binding Domain in insect cells and purify them for functional and structural studies.
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crystal structure of the rar gamma Ligand Binding Domain bound to all trans retinoic acid
Nature, 1995Co-Authors: Jeanpaul Renaud, Marc Ruff, Pierre Chambon, Hinrich Gronemeyer, Natacha Rochel, Valeria Vivat, Dino MorasAbstract:The 2.0-A crystal structure of the Ligand-Binding Domain (LBD) of the human retinoic acid receptor (RAR)-γ bound to all-trans retinoic acid reveals the Ligand-Binding interactions and suggests an electrostatic guidance mechanism. The overall fold is similar to that of the human RXR-α apo-LBD, except for the carboxy-terminal part which folds back towards the LBD core, contributing to the hydrophobic Ligand pocket and 'sealing' its entry site. We propose a 'mouse trap' mechanism whereby a Ligand-induced conformational transition repositions the amphi-pathic α-helix of the AF-2 activating Domain and forms a transcriptionally active receptor.
Eric Gouaux - One of the best experts on this subject based on the ideXlab platform.
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Probing the Ligand Binding Domain of the GluR2 receptor by proteolysis and deletion mutagenesis defines Domain boundaries and yields a crystallizable construct
Protein science : a publication of the Protein Society, 1998Co-Authors: Guo Qiang Chen, Yu Sun, Rongsheng Jin, Eric GouauxAbstract:Ionotropic glutamate receptors constitute an important family of Ligand-gated ion channels for which there is little biochemical or structural data. Here we probe the Domain structure and boundaries of the Ligand Binding Domain of the AMPA-sensitive GluR2 receptor by limited proteolysis and deletion mutagenesis. To identify the proteolytic fragments, Maldi mass spectrometry and N-terminal amino acid sequencing were employed. Trypsin digestion of HS1S2 (Chen GQ, Gouaux E. 1997. Proc Natl Acad Sci USA 94:13431-13436) in the presence and absence of glutamate showed that the Ligand stabilized the S1 and S2 fragments against complete digestion. Using limited proteolysis and multiple sequence alignments of glutamate receptors as guides, nine constructs were made, folded, and screened for Ligand Binding activity. From this screen, the S1S21 construct proved to be trypsin- and chymotrypsin-resistant, stable to storage at 4 degrees C, and amenable to three-dimensional crystal formation. The HS1S21 variant was readily prepared on a large scale, the His tag was easily removed by trypsin, and crystals were produced that diffracted to beyond 1.5 A resolution. These experiments, for the first time, pave the way to economical overproduction of the Ligand Binding Domains of glutamate receptors and more accurately map the boundaries of the Ligand Binding Domain.
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Overexpression of a glutamate receptor (GluR2) Ligand Binding Domain in Escherichia coli: Application of a novel protein folding screen
Proceedings of the National Academy of Sciences of the United States of America, 1997Co-Authors: Guo Qiang Chen, Eric GouauxAbstract:Expression of the S1S2 Ligand Binding Domain [Kuusinen, A., Arvola, M. & Keinanen, K. (1995) EMBO J. 14, 6327–6332] of the rat α-amino-3-hydroxy-5-methylisoxazole-4-propionic acid-selective glutamate receptor GluR2 in Escherichia coli under control of a T7 promoter leads to production of >100 mg/liter of histidine-tagged S1S2 protein (HS1S2) in the form of inclusion bodies. Using a novel fractional factorial folding screen and a rational, step-by-step approach, multiple conditions were determined for the folding of the HS1S2 α-amino-3-hydroxy-5-methylisoxazole-4-propionic acid Binding Domain. Characterization of the HS1S2 Ligand Binding Domain showed that it is water-soluble, monomeric, has significant secondary structure, and is sensitive to trypsinolysis at sites close to the beginning of the putative transmembrane regions. Application of a fractional factorial folding screen to other proteins may provide a useful means to evaluate E. coli as an economical and convenient expression host.
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overexpression of a glutamate receptor glur2 Ligand Binding Domain in escherichia coli application of a novel protein folding screen
Proceedings of the National Academy of Sciences of the United States of America, 1997Co-Authors: Guo Qiang Chen, Eric GouauxAbstract:Expression of the S1S2 Ligand Binding Domain [Kuusinen, A., Arvola, M. & Keinanen, K. (1995) EMBO J. 14, 6327–6332] of the rat α-amino-3-hydroxy-5-methylisoxazole-4-propionic acid-selective glutamate receptor GluR2 in Escherichia coli under control of a T7 promoter leads to production of >100 mg/liter of histidine-tagged S1S2 protein (HS1S2) in the form of inclusion bodies. Using a novel fractional factorial folding screen and a rational, step-by-step approach, multiple conditions were determined for the folding of the HS1S2 α-amino-3-hydroxy-5-methylisoxazole-4-propionic acid Binding Domain. Characterization of the HS1S2 Ligand Binding Domain showed that it is water-soluble, monomeric, has significant secondary structure, and is sensitive to trypsinolysis at sites close to the beginning of the putative transmembrane regions. Application of a fractional factorial folding screen to other proteins may provide a useful means to evaluate E. coli as an economical and convenient expression host.
Anu Rambhadran - One of the best experts on this subject based on the ideXlab platform.
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LRET Investigations of Conformational Changes in the Ligand Binding Domain of a Functional AMPA Receptor
Biochemistry, 2008Co-Authors: Jennifer M. Reingle Gonzalez, Anu Rambhadran, Vasanthi JayaramanAbstract:The structural investigations using the soluble Ligand Binding Domain of the AMPA subtype of the glutamate receptor have provided invaluable insight into the mechanistic pathway by which agonist Binding to this extracellular Domain mediates the formation of cation-selective channels in this protein. These structures, however, are in the absence of the transmembrane segments, the primary functional component of the protein. Here, we have used a modified luminescence resonance energy transfer based method to obtain distance changes due to agonist Binding in the Ligand Binding Domain in the presence of the transmembrane segments. These distance changes show that the cleft closure conformational change observed in the isolated Ligand Binding Domain upon Binding agonist is conserved in the receptor with the channel segments, thus establishing that the isolated Ligand Binding Domain is a good model of the Domain in the receptor containing the transmembrane segments.
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Luminescence Resonance Energy Transfer Investigation of Conformational Changes in the Ligand Binding Domain of a Kainate Receptor
The Journal of biological chemistry, 2008Co-Authors: Anu Rambhadran, Vasanthi JayaramanAbstract:The apo state structure of the isolated Ligand Binding Domain of the GluR6 subunit and the conformational changes induced by agonist Binding to this protein have been investigated by luminescence resonance energy transfer (LRET) measurements. The LRET-based distances show that agonist Binding induces cleft closure, and the extent of cleft closure is proportional to the extent of activation over a wide range of activations, thus establishing that the cleft closure conformational change is one of the mechanisms by which the agonist mediates receptor activation. The LRET distances also provide insight into the apo state structure, for which there is currently no crystal structure available. The distance change between the glutamate-bound state and the apo state is similar to that observed between the glutamate-bound and antagonist UBP-310-bound form of the GluR5 Ligand Binding Domain, indicating that the cleft for the apo state of the GluR6 Ligand Binding Domain should be similar to the UBP-310-bound form of GluR5. This observation implies that te apo state of GluR6 undergoes a cleft closure of 29–30° upon Binding full agonists, one of the largest observed in the glutamate receptor family.
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Luminescence Resonance Energy Transfer Investigation of Conformational Changes in the Ligand Binding Domain of a
2008Co-Authors: Anu Rambhadran, Vasanthi JayaramanAbstract:The apo state structure of the isolated Ligand Binding Domain of the GluR6 subunit and the conformational changes induced by agonist Binding to this protein have been investigated by luminescence resonance energy transfer (LRET) measurements. The LRET-based distances show that agonist Binding induces cleft closure, and the extent of cleft closure is proportional to the extent of activation over a wide range of activations, thus establishing that the cleft closure conformational change is one of the mechanisms by which the agonist mediates receptor activation. The LRET distances also provide insight into the apo state structure, for which there is currently no crystal structure available. The distance change between the glutamate-bound state and the apo state is similar to that observed between the glutamate-bound and antagonist UBP-310-bound form of the GluR5 Ligand Binding Domain, indicating that the cleft for the apo state of the GluR6 Ligand Binding Domain should be similar to the UBP-310bound form of GluR5. This observation implies that the apo state of GluR6 undergoes a cleft closure of 29 –30° upon Binding full agonists, one of the largest observed in the glutamate receptor family.