RESUMEN
N-methyl-D-aspartate receptors (NMDARs) play essential roles in memory formation, neuronal plasticity, and brain development, with their dysfunction linked to a range of disorders from ischemia to schizophrenia. Zinc and pH are physiological allosteric modulators of NMDARs, with GluN2A-containing receptors inhibited by nanomolar concentrations of divalent zinc and by excursions to low pH. Despite the widespread importance of zinc and proton modulation of NMDARs, the molecular mechanism by which these ions modulate receptor activity has proven elusive. Here, we use cryoelectron microscopy to elucidate the structure of the GluN1/GluN2A NMDAR in a large ensemble of conformations under a range of physiologically relevant zinc and proton concentrations. We show how zinc binding to the amino terminal domain elicits structural changes that are transduced though the ligand-binding domain and result in constriction of the ion channel gate.
Asunto(s)
Complejos Multiproteicos/química , Protones , Receptores de N-Metil-D-Aspartato/química , Zinc/química , Regulación Alostérica , Animales , Microscopía por Crioelectrón , Concentración de Iones de Hidrógeno , Complejos Multiproteicos/genética , Complejos Multiproteicos/metabolismo , Complejos Multiproteicos/ultraestructura , Dominios Proteicos , Ratas , Receptores de N-Metil-D-Aspartato/genética , Receptores de N-Metil-D-Aspartato/metabolismo , Células Sf9 , Spodoptera , Zinc/metabolismoRESUMEN
Perception of heat or cold in higher organisms is mediated by specialized ion channels whose gating is exquisitely sensitive to temperature. The physicochemical underpinnings of this temperature-sensitive gating have proven difficult to parse. Here, we took a bottom-up protein design approach and rationally engineered ion channels to activate in response to thermal stimuli. By varying amino acid polarities at sites undergoing state-dependent changes in solvation, we were able to systematically confer temperature sensitivity to a canonical voltage-gated ion channel. Our results imply that the specific heat capacity change during channel gating is a major determinant of thermosensitive gating. We also show that reduction of gating charges amplifies temperature sensitivity of designer channels, which accounts for low-voltage sensitivity in all known temperature-gated ion channels. These emerging principles suggest a plausible molecular mechanism for temperature-dependent gating that reconcile how ion channels with an overall conserved transmembrane architecture may exhibit a wide range of temperature-sensing phenotypes. :
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Canales Iónicos/química , Canales Iónicos/metabolismo , Ingeniería de Proteínas , Animales , Humanos , Interacciones Hidrofóbicas e Hidrofílicas , Modelos Moleculares , Ratas , Temperatura , TermodinámicaRESUMEN
Electrical activity in the brain and heart depends on rhythmic generation of action potentials by pacemaker ion channels (HCN) whose activity is regulated by cAMP binding1. Previous work has uncovered evidence for both positive and negative cooperativity in cAMP binding2,3, but such bulk measurements suffer from limited parameter resolution. Efforts to eliminate this ambiguity using single-molecule techniques have been hampered by the inability to directly monitor binding of individual ligand molecules to membrane receptors at physiological concentrations. Here we overcome these challenges using nanophotonic zero-mode waveguides4 to directly resolve binding dynamics of individual ligands to multimeric HCN1 and HCN2 ion channels. We show that cAMP binds independently to all four subunits when the pore is closed, despite a subsequent conformational isomerization to a flip state at each site. The different dynamics in binding and isomerization are likely to underlie physiologically distinct responses of each isoform to cAMP5 and provide direct validation of the ligand-induced flip-state model6-9. This approach for observing stepwise binding in multimeric proteins at physiologically relevant concentrations can directly probe binding allostery at single-molecule resolution in other intact membrane proteins and receptors.
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AMP Cíclico/metabolismo , Canales Regulados por Nucleótidos Cíclicos Activados por Hiperpolarización/metabolismo , Activación del Canal Iónico , Células HEK293 , Humanos , Ligandos , Unión Proteica , Ingeniería de Proteínas , Isoformas de Proteínas , Multimerización de Proteína , Imagen Individual de MoléculaRESUMEN
Although many redox signaling molecules are present at low concentrations, typically ranging from micromolar to submicromolar levels, they often play essential roles in a wide range of biological pathways and disease mechanisms. However, accurately measuring low-abundant analytes has been a significant challenge due to the lack of sensitivity and quantitative capability of existing measurement methods. In this study, we introduced a novel chemically induced amplifiable system for quantifying low-abundance redox signaling molecules in living cells. We utilized H2O2 as a proof-of-concept analyte and developed a probe that quantifies cellular peroxide levels by combining the NanoBiT system with androgen receptor dimerization as a reporting mechanism. Our system demonstrated a highly sensitive response to cellular peroxide changes induced both endogenously and exogenously. Furthermore, the system can be adapted for the quantification of other signaling molecules if provided with suitable probing chemistry.
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Peróxido de Hidrógeno , Receptores Androgénicos , Peróxido de Hidrógeno/análisis , Peróxido de Hidrógeno/química , Humanos , Receptores Androgénicos/metabolismo , Oxidación-ReducciónRESUMEN
In the scope of targeted protein degradation (TPD), proteolysis-targeting chimeras (PROTACs), leveraging the ubiquitin-proteasome system, have been extensively studied. However, they are limited to the degradation of soluble and membrane proteins, excluding the aggregated and extracellular proteins and dysfunctional organelles. As an alternative protein degradation pathway, lysosomes serve as a feasible tool for accessing these untouched proteins and/or organelles by proteosomes. Here, we focus on reviewing the emerging lysosome-mediated TPD, such as AUTAC, ATTEC, AUTOTAC, LYTAC, and MoDE-A. Intracellular targets, such as soluble and aggregated proteins and organelles, can be degraded via the autophagy-lysosome pathway. Extracellular targets, such as membrane proteins, and secreted extracellular proteins can be degraded via the endosome-lysosome pathway. In addition, we summarize the mechanism and regulation of autophagy, available methods and assays for monitoring the autophagy process, and the recently developed chemical probes for perturbing the autophagy pathways.
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Complejo de la Endopetidasa Proteasomal , Ubiquitina , Proteolisis , Complejo de la Endopetidasa Proteasomal/metabolismo , Ubiquitina/metabolismo , Autofagia/fisiología , Proteínas de la Membrana/metabolismo , Lisosomas/metabolismoRESUMEN
The introduction of noncanonical amino acids into proteins and peptides has been of great interest for many years and has facilitated the detailed study of peptide/protein structure and mechanism. In addition to numerous nonproteinogenic α-l-amino acids, bacterial ribosome modification has provided the wherewithal to enable the synthesis of peptides and proteins with a much greater range of structural diversity, as has the use of endogenous bacterial proteins in reconstituted protein synthesizing systems. In a recent report, elongation factor P (EF-P), putatively essential for enabling the incorporation of contiguous proline residues into proteins, was shown to facilitate the introduction of an N-methylated amino acid in addition to proline. This finding prompted us to investigate the properties of this protein factor with a broad variety of structurally diverse amino acid analogues using an optimized suppressor tRNAPro that we designed. While these analogues can generally be incorporated into proteins only in systems containing modified ribosomes specifically selected for their incorporation, we found that EF-P could significantly enhance their incorporation into model protein dihydrofolate reductase using wild-type ribosomes. Plausibly, the increased yields observed in the presence of structurally diverse amino acid analogues may result from the formation of a stabilized ribosomal complex in the presence of EF-P that provides more favorable conditions for peptide bond formation. This finding should enable the facile incorporation of a much broader structural variety of amino acid analogues into proteins and peptides using native ribosomes.
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Aminoácidos , Escherichia coli , Aminoácidos/química , Escherichia coli/metabolismo , Tetrahidrofolato Deshidrogenasa/metabolismo , Factores de Elongación de Péptidos/metabolismo , Péptidos/química , Prolina/metabolismoRESUMEN
Here we present a virtual docking screen of 1648 commercially available covalent fragments, and identified covalent inhibitors of cysteine protease cathepsin L. These inhibitors did not inhibit closely related protease cathepsin B. Thus, we have established virtual docking of covalent fragments as an approach to discover covalent enzyme inhibitors.
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Catepsina L/antagonistas & inhibidores , Inhibidores de Cisteína Proteinasa/farmacología , Descubrimiento de Drogas , Simulación del Acoplamiento Molecular , Catepsina L/metabolismo , Inhibidores de Cisteína Proteinasa/síntesis química , Inhibidores de Cisteína Proteinasa/química , Relación Dosis-Respuesta a Droga , Evaluación Preclínica de Medicamentos , Humanos , Estructura Molecular , Relación Estructura-ActividadRESUMEN
Friedreich's ataxia (FRDA) is an autosomal recessive neurodegenerative disorder resulting from reduced expression of the protein frataxin (FXN). Although its function is not fully understood, frataxin appears to help assemble iron sulfur clusters; these are critical for the function of many proteins, including those needed for mitochondrial energy production. Finding ways to increase FXN levels has been a major therapeutic strategy for this disease. Previously, we described a novel series of methylene violet analogues and their structural optimization as potential therapeutic agents for neurodegenerative and mitochondrial disorders. Presently, a series of methylene blue analogues has been synthesized and characterized for their in vitro biochemical and biological properties in cultured Friedreich's ataxia lymphocytes. Favorable methylene blue analogues were shown to increase frataxin levels and mitochondrial biogenesis, and to improve aconitase activity. The analogues were found to be good ROS scavengers, and able to protect cultured FRDA lymphocytes from oxidative stress resulting from inhibition of complex I and from glutathione depletion. The analogues also preserved mitochondrial membrane potential and augmented ATP production. Our results suggest that analogue 5, emerging from the initial structure of the parent compound methylene blue (MB), represents a promising lead structure and lacks the cytotoxicity associated with the parent compound MB.
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Proteínas de Unión a Hierro/metabolismo , Azul de Metileno/análogos & derivados , Mitocondrias/efectos de los fármacos , Adenosina Trifosfato/metabolismo , Línea Celular , Supervivencia Celular/efectos de los fármacos , Complejo I de Transporte de Electrón/metabolismo , Ataxia de Friedreich/metabolismo , Ataxia de Friedreich/patología , Glutatión/metabolismo , Humanos , Linfocitos/citología , Linfocitos/efectos de los fármacos , Linfocitos/metabolismo , Potencial de la Membrana Mitocondrial/efectos de los fármacos , Azul de Metileno/farmacología , Mitocondrias/metabolismo , Estrés Oxidativo/efectos de los fármacos , Especies Reactivas de Oxígeno/metabolismo , FrataxinaRESUMEN
In an effort to identify methylene blue analogues having improved antioxidant activity, a series of new methylene violet analogues have been designed and synthesized. The analogues were prepared following a synthetic route that is more efficient than the previously reported methods, both in terms of yield and purity of the final products. The route involves the Smiles rearrangement as one of the crucial steps. Smiles rearrangement of suitably substituted diphenyl sulfide intermediates afforded the corresponding phenothiazine analogues in high yields, which were subsequently converted to the final products. The methylene violet analogues were evaluated for their ability to preserve mitochondrial function in Friedreich's ataxia (FRDA) lymphocytes. The analogues were shown to be efficient ROS scavengers, and able to protect cultured FRDA lymphocytes from oxidative stress resulting from inhibition of complex I. The analogues also preserved mitochondrial membrane potential and augmented ATP production. The analogues were found to be better antioxidants than the parent compounds methylene blue and methylene violet.
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Mitocondrias/efectos de los fármacos , Fenotiazinas/farmacología , Adenosina Trifosfato/biosíntesis , Células Cultivadas , Humanos , Interacciones Hidrofóbicas e Hidrofílicas , Linfocitos/efectos de los fármacos , Linfocitos/metabolismo , Potencial de la Membrana Mitocondrial/efectos de los fármacos , Mitocondrias/metabolismo , Estrés Oxidativo/efectos de los fármacos , Fenotiazinas/síntesis química , Fenotiazinas/química , Especies Reactivas de Oxígeno/metabolismoRESUMEN
Described herein are the synthesis and photophysical characterization of a library of aryl-substituted oxazole- and thiazole-based dipeptidomimetic analogues, and their incorporation into position 66 of green fluorescent protein (GFP) in lieu of the natural fluorophore. These fluorescent analogues resemble the fluorophore formed naturally by GFP. As anticipated, the photophysical properties of the analogues varied as a function of the substituents at the para position of the phenyl ring. The fluorescence emission wavelength maxima of compounds in the library varied from â¼365 nm (near-UV region) to â¼490 nm (visible region). The compounds also exhibited a large range of quantum yields (0.01-0.92). The analogues were used to activate a suppressor tRNACUA and were incorporated into position 66 of GFP using an in vitro protein biosynthesizing system that employed engineered ribosomes selected for their ability to incorporate dipeptides. Four analogues with interesting photophysical properties and reasonable suppression yields were chosen, and the fluorescent proteins (FPs) containing these fluorophores were prepared on a larger scale for more detailed study. When the FPs were compared with the respective aminoacyl-tRNAs and the actual dipeptide analogues, the FPs exhibited significantly enhanced fluorescence intensities at the same concentrations. Part of this was shown to be due to the presence of the fluorophores as an intrinsic element of the protein backbone. There were also characteristic shifts in the emission maxima, indicating the environmental sensitivity of these probes. Acridon-2-ylalanine and oxazole 1a were incorporated into positions 39 and 66 of GFP, respectively, and were shown to form an efficient Förster resonance energy transfer (FRET) pair, demonstrating that the analogues can be used as FRET probes.
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Dipéptidos/metabolismo , Escherichia coli/metabolismo , Fluorescencia , Peptidomiméticos/síntesis química , Peptidomiméticos/metabolismo , ARN de Transferencia/metabolismo , Ribosomas/metabolismo , Dipéptidos/síntesis química , Transferencia Resonante de Energía de Fluorescencia , Colorantes Fluorescentes , Proteínas Fluorescentes Verdes , Humanos , Modelos Moleculares , Estructura Molecular , Biosíntesis de ProteínasRESUMEN
In an earlier study, ß³-puromycin was used for the selection of modified ribosomes, which were utilized for the incorporation of five different ß-amino acids into Escherichia coli dihydrofolate reductase (DHFR). The selected ribosomes were able to incorporate structurally disparate ß-amino acids into DHFR, in spite of the use of a single puromycin for the selection of the individual clones. In this study, we examine the extent to which the structure of the ß³-puromycin employed for ribosome selection influences the regio- and stereochemical preferences of the modified ribosomes during protein synthesis; the mechanistic probe was a single suppressor tRNA(CUA) activated with each of four methyl-ß-alanine isomers (1-4). The modified ribosomes were found to incorporate each of the four isomeric methyl-ß-alanines into DHFR but exhibited a preference for incorporation of 3(S)-methyl-ß-alanine (ß-mAla; 4), i.e., the isomer having the same regio- and stereochemistry as the O-methylated ß-tyrosine moiety of ß³-puromycin. Also conducted were a selection of clones that are responsive to ß²-puromycin and a demonstration of reversal of the regio- and stereochemical preferences of these clones during protein synthesis. These results were incorporated into a structural model of the modified regions of 23S rRNA, which included in silico prediction of a H-bonding network. Finally, it was demonstrated that incorporation of 3(S)-methyl-ß-alanine (ß-mAla; 4) into a short α-helical region of the nucleic acid binding domain of hnRNP LL significantly stabilized the helix without affecting its DNA binding properties.
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Alanina/análogos & derivados , Proteínas de Escherichia coli/biosíntesis , Ribonucleoproteína Heterogénea-Nuclear Grupo L/biosíntesis , Modelos Moleculares , ARN Bacteriano/metabolismo , ARN Ribosómico/metabolismo , Ribosomas/metabolismo , Tetrahidrofolato Deshidrogenasa/biosíntesis , Alanina/química , Alanina/metabolismo , Escherichia coli/enzimología , Escherichia coli/metabolismo , Proteínas de Escherichia coli/química , Ribonucleoproteína Heterogénea-Nuclear Grupo L/química , Ribonucleoproteína Heterogénea-Nuclear Grupo L/genética , Humanos , Enlace de Hidrógeno , Simulación de Dinámica Molecular , Proteínas Mutantes/biosíntesis , Proteínas Mutantes/química , Proteínas Mutantes/genética , Motivos de Nucleótidos , Peptidil Transferasas/genética , Peptidil Transferasas/metabolismo , Conformación Proteica , Estabilidad Proteica , Puromicina/análogos & derivados , Puromicina/química , Puromicina/metabolismo , ARN Bacteriano/química , ARN Ribosómico/química , ARN Ribosómico 23S/química , ARN Ribosómico 23S/metabolismo , Aminoacil-ARN de Transferencia/química , Aminoacil-ARN de Transferencia/metabolismo , Proteínas Recombinantes/biosíntesis , Proteínas Recombinantes/química , Estereoisomerismo , Especificidad por Sustrato , Tetrahidrofolato Deshidrogenasa/químicaRESUMEN
Plasmids containing 23S rRNA randomized at positions 2057-2063 and 2502-2507 were introduced into Escherichia coli, affording a library of clones which produced modified ribosomes in addition to the pre-existing wild-type ribosomes. These clones were screened with a derivative of puromycin, a natural product which acts as an analogue of the 3'-end of aminoacyl-tRNA and terminates protein synthesis by accepting the growing polypeptide chain, thereby killing bacterial cells. The puromycin derivative in this study contained the dipeptide p-methoxyphenylalanylglycine, implying the ability of the modified ribosomes in clones sensitive to this puromycin analogue to recognize dipeptides. Several clones inhibited by the puromycin derivative were used to make S-30 preparations, and some of these were shown to support the incorporation of dipeptides into proteins. The four incorporated species included two dipeptides (Gly-Phe (2) and Phe-Gly (3)), as well as a thiolated dipeptide analogue (4) and a fluorescent oxazole (5) having amine and carboxyl groups approximately the same distance apart as in a normal dipeptide. A protein containing both thiolated dipeptide 4 and a 7-methoxycoumarin fluorophore was found to undergo fluorescence quenching. Introduction of the oxazole fluorophore 5 into dihydrofolate reductase or green fluorescent protein resulted in quite strong enhancement of its fluorescence emission, and the basis for this enhancement was studied. The aggregate results demonstrate the feasibility of incorporating dipeptides as a single ribosomal event, and illustrate the lack of recognition of the central peptide bond in the dipeptide, potentially enabling the incorporation of a broad variety of structural analogues.
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Dipéptidos/química , Dipéptidos/metabolismo , Proteínas Fluorescentes Verdes/metabolismo , Ribosomas/metabolismo , Tetrahidrofolato Deshidrogenasa/metabolismo , Proteínas Fluorescentes Verdes/química , Modelos Moleculares , Conformación Proteica , Tetrahidrofolato Deshidrogenasa/químicaRESUMEN
The synthesis and incorporation into position 66 of green fluorescent protein (GFP) by in vitro protein translation of novel oxazole and thiazole based dipeptidomimetics are described. The compounds may be regarded as GFP chromophore analogues, and are strongly fluorescent. An α-amido-ß-ketoester intermediate was obtained via bisacylation of a protected glycine. The intermediate underwent dehydrative cyclization to afford the 1,3-oxazole and was treated with Lawesson's reagent to furnish the 1,3-thiazole. When these fluorophores were introduced into position 66 of GFP in place of Tyr66, the resulting GFP analogues exhibited fluorescence emission several-fold greater than wild-type GFP; the emission was also shifted to shorter wavelength. It may be noted that compared to the typical fluorophores formed in the natural and modified fluorescent proteins, the oxazole and thiazole fluorophores are completely stable and do not require activation by posttranslational modification to exhibit fluorescence.
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Colorantes Fluorescentes/síntesis química , Proteínas Fluorescentes Verdes/química , Proteínas Fluorescentes Verdes/metabolismo , Peptidomiméticos/síntesis química , Peptidomiméticos/metabolismo , Ribosomas/metabolismo , Fluorescencia , Colorantes Fluorescentes/química , Colorantes Fluorescentes/metabolismo , Estructura Molecular , Peptidomiméticos/químicaRESUMEN
A series of Chk1 degraders were designed and synthesized. The degraders were developed through the conjugation of a promiscuous kinase binder and thalidomide. One of the degraders PROTAC-2 was able to decrease Chk1 levels in a concentration-dependent manner in A375 cells. The developed probes can be useful for the development of selective and more potent Chk1 degraders.
RESUMEN
Steroid receptor coactivator 3 (SRC-3) is a critical mediator of many intracellular signaling pathways that are crucial for cancer proliferation and metastasis. In this study, we performed structure-activity relationship exploration and drug-like optimization of the hit compound SI-2, guided by in vitro/in vivo metabolism studies and cytotoxicity assays. Our efforts led to the discovery of two lead compounds, SI-10 and SI-12. Both compounds exhibit potent cytotoxicity against a panel of human cancer cell lines and demonstrate acceptable pharmacokinetic properties. A biotinylated estrogen response element pull-down assay demonstrated that SI-12 could disrupt the recruitment of SRC-3 and p300 in the estrogen receptor complex. Importantly, SI-10 and SI-12 significantly inhibited tumor growth and metastasis in vivo without appreciable acute toxicity. These results demonstrate the potential of SI-10 and SI-12 as drug candidates for cancer therapy, given their potent SRC-3 inhibition and promising pharmacokinetic and toxicity profiles.
Asunto(s)
Antineoplásicos , Neoplasias , Humanos , Coactivador 3 de Receptor Nuclear/metabolismo , Línea Celular , Relación Estructura-Actividad , Transducción de Señal , Proliferación Celular , Línea Celular Tumoral , Antineoplásicos/farmacologíaRESUMEN
Ribosomes containing modifications in three regions of 23S rRNA, all of which are in proximity to the ribosomal peptidyltransferase center (PTC), were utilized previously as a source of S-30 preparations for in vitro protein biosynthesis experiments. When utilized in the presence of mRNAs containing UAG codons at predetermined positions+ß-alanyl-tRNA(CUA), the modified ribosomes produced enhanced levels of full length proteins via UAG codon suppression. In the present study, these earlier results have been extended by the use of substituted ß-amino acids, and direct evidence for ß-amino acid incorporation is provided. Presently, five of the clones having modified ribosomes are used in experiments employing four substituted ß-amino acids, including α-methyl-ß-alanine, ß,ß-dimethyl-ß-alanine, ß-phenylalanine, and ß-(p-bromophenyl)alanine. The ß-amino acids were incorporated into three different positions (10, 18 and 49) of Escherichia coli dihydrofolate reductase (DHFR) and their efficiencies of suppression of the UAG codons were compared with those of ß-alanine and representative α-l-amino acids. The isolated proteins containing the modified ß-amino acids were subjected to proteolytic digestion, and the derived fragments were characterized by mass spectrometry, establishing that the ß-amino acids had been incorporated into DHFR, and that they were present exclusively in the anticipated peptide fragments. DHFR contains glutamic acid in position 17, and it has been shown previously that Glu-C endoproteinase can hydrolyze DHFR between amino acids residues 17 and 18. The incorporation of ß,ß-dimethyl-ß-alanine into position 18 of DHFR prevented this cleavage, providing further evidence for the position of incorporation of the ß-amino acid.
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Aminoácidos/química , Ribosomas/metabolismo , Tetrahidrofolato Deshidrogenasa/química , Secuencia de Aminoácidos , Aminoácidos/metabolismo , Escherichia coli/enzimología , Datos de Secuencia Molecular , Péptidos/análisis , ARN Ribosómico 23S/química , ARN Ribosómico 23S/metabolismo , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción , Tetrahidrofolato Deshidrogenasa/metabolismo , beta-Alanina/química , beta-Alanina/metabolismoRESUMEN
Cooperative interactions mediate information transfer between structural domains of a protein molecule and are major determinants of protein function and modulation. The prevalent theories to understand the thermodynamic origins of cooperativity have been developed to reproduce the complex behavior of a global thermodynamic observable such as ligand binding or enzyme activity. However, in most cases the measurement of a single global observable cannot uniquely define all the terms that fully describe the energetics of the system. Here we establish a theoretical groundwork for analyzing protein thermodynamics using site-specific information. Our treatment involves extracting a site-specific parameter (defined as χ value) associated with a structural unit. We demonstrate that, under limiting conditions, the χ value is related to the direct interaction terms associated with the structural unit under observation and its intrinsic activation energy. We also introduce a site-specific interaction energy term (χ(diff)) that is a function of the direct interaction energy of that site with every other site in the system. When combined with site-directed mutagenesis and other molecular level perturbations, analyses of χ values of site-specific observables may provide valuable insights into protein thermodynamics and structure.
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Enzimas/química , Modelos Químicos , Enzimas/metabolismo , Ligandos , TermodinámicaRESUMEN
The sperm-specific sodium hydrogen exchanger, SLC9C1, underlies hyperpolarization and cyclic nucleotide stimulated proton fluxes across sperm membranes and regulates their hyperactivated motility. SLC9C1 is the first known instance of an ion transporter that uses a canonical voltage-sensing domain (VSD) and an evolutionarily conserved cyclic nucleotide binding domain (CNBD) to influence the dynamics of its ion-exchange domain (ED). The structural organization of this 'tripartite transporter' and the mechanisms whereby it integrates physical (membrane voltage) and chemical (cyclic nucleotide) cues are unknown. In this study, we use single particle cryo-electron microscopy to determine structures of a metazoan SLC9C1 in different conformational states. We find that the three structural domains are uniquely organized around a distinct ring-shaped scaffold that we call the 'allosteric ring domain' or ARD. The ARD undergoes coupled proton-dependent rearrangements with the ED and acts as a 'signaling hub' enabling allosteric communication between the key functional modules of sp9C1. We demonstrate that binding of cAMP causes large conformational changes in the cytoplasmic domains and disrupts key ARD-linked interfaces. We propose that these structural changes rescue the transmembrane domains from an auto-inhibited state and facilitate their functional dynamics. Our study provides a structural framework to understand and further probe electrochemical linkage in SLC9C1.
RESUMEN
Multi-modal regulation of Slo1 channels by membrane voltage, intracellular calcium, and auxiliary subunits enables its pleiotropic physiological functions. Our understanding of how voltage impacts Slo1 conformational dynamics and the mechanisms by which auxiliary subunits, particularly of the LRRC (Leucine Rich Repeat containing) family of proteins, modulate its voltage gating remain unresolved. Here, we used single particle cryo-electron microscopy to determine structures of human Slo1 mutants which functionally stabilize the closed pore (F315A) or the activated voltage-sensor (R207A). Our structures, obtained under calcium-free conditions, reveal that a key step in voltage-sensing by Slo1 involves a rotameric flip of the voltage-sensing charges (R210 and R213) moving them by â¼6 Å across a hydrophobic gasket. Next we obtained reconstructions of a complex of human Slo1 with the human LRRC26 (γ1) subunit in absence of calcium. Together with extensive biochemical tests, we show that the extracellular domains of γ1 form a ring of interlocked dominos that stabilizes the quaternary assembly of the complex and biases Slo1:γ1 assembly towards high stoichiometric complexes. The transmembrane helix of γ1 is kinked and tightly packed against the Slo1 voltage-sensor. We hypothesize that γ1 subunits exert relatively small effects on early steps in voltage-gating but structurally stabilize non-S4 helices of Slo1 voltage-sensor which energetically facilitate conformational rearrangements that occur late in voltage stimulated transitions.
RESUMEN
AIM: This study aimsâ to report preclinical validation, and the first clinical treatment of total bone marrow irradiation (TMI) and total bone marrow and lymph nodal irradiation (TMLI) using Volumetric modulated arc therapy in Halcyon-E ring gantry linear accelerator. Preclinical validation includes simulation, planning, patient-specific QA, and dry run. MATERIAL AND METHOD: Four patients, two female and two male, with body weights of 116 kg, 52 kg, 64 kg, and 62 kg; with two with chronic myeloid leukemia, one each with acute lymphoblastic leukemia and acute myeloid leukemia (AML) were simulated and planned for TMI/TMLI. Patients were immobilized with a full-body vacuum bag. Head first supine (HFS) and Feet first supine (FFS) CT scans were acquired from head to knee and knee to toe. Planning target volume (PTV) was created with a uniform margin of 6 mm over the total bone marrow/bone marrow + lymph nodes. HFS and FFS PTVs were optimized independently using 6MV unflatten energy for 12 Gy in 6 fractions. Plans were merged to create the resultant dose distribution using a junction bias dose matching technique. The total number of isocenters was ≤ 10 per CT set, and two to four full arcs were used for each isocenter. A junction dose gradient technique was used for dose feathering between arcs between adjacent isocenters. RESULT: Only one female patient diagnosed as AML received the TMLI treatment, while the other three patients dropped out due to clinical complications and comorbidities that developed in the time between simulation and treatment. The result presented has been averaged over all four patients. For PTV, 95% dose was normalised to 95% volume, PTV_V107% receiving 3.3 ± 3.1%. Total lung mean and V12Gy were 1048.6 ± 107.1 cGy and 19.5 ± 12.1%. Maximum lens doses were 489.5 ± 35.5 cGy (left: L) and 497 ± 69.2 cGy (right: R). The mean cardiac and bilateral kidney doses were 921.75 ± 89.2 cGy, 917.9 ± 63.2 cGy (L), and 805.9 ± 9.7 cGy (R). Average Monitor Unit was 7738.25 ± 1056.6. The median number of isocenters was 17(HFS+FFS), average MU/Dose (cGy) ratio per isocenter was 2.28 ± 0.3. CONCLUSION: Halcyon-E ring gantry linear accelerator capable of planning and delivering TMI/TMLI.ââ.