RESUMEN
The mitochondrial calcium uniporter (mtCU) is a multicomponent Ca 2+ -specific channel that imparts mitochondria with the capacity to sense the cytosolic calcium signals. The metazoan mtCU comprises the pore-forming subunit MCU and the essential regulator EMRE, arranged in a tetrameric channel complex, and the Ca 2+ sensing peripheral proteins MICU1-3. The mechanism of mitochondrial Ca 2+ uptake by mtCU and its regulation is poorly understood. Our analysis of MCU structure and sequence conservation, combined with molecular dynamics simulations, mutagenesis, and functional studies, led us to conclude that the Ca 2+ conductance of MCU is driven by a ligand-relay mechanism, which depends on stochastic structural fluctuations in the conserved DxxE sequence. In the tetrameric structure of MCU, the four glutamate side chains of DxxE (the E-ring) chelate Ca 2+ directly in a high-affinity complex (site 1), which blocks the channel. The four glutamates can also switch to a hydrogen bond-mediated interaction with an incoming hydrated Ca 2+ transiently sequestered within the D-ring of DxxE (site 2), thus releasing the Ca 2+ bound at site 1. This process depends critically on the structural flexibility of DxxE imparted by the adjacent invariant Pro residue. Our results suggest that the activity of the uniporter can be regulated through the modulation of local structural dynamics. A preliminary account of this work was presented at the 67 th Annual Meeting of the Biophysical Society in San Diego, CA, February 18-22, 2023.
RESUMEN
We describe the case of identical twin boys who presented with low body weight despite excessive caloric intake. An evaluation of their fibroblasts showed elevated oxygen consumption and decreased mitochondrial membrane potential. Exome analysis revealed a de novo heterozygous variant in ATP5F1B, which encodes the ß subunit of mitochondrial ATP synthase (also called complex V). In yeast, mutations affecting the same region loosen coupling between the proton motive force and ATP synthesis, resulting in high rates of mitochondrial respiration. Expression of the mutant allele in human cell lines recapitulates this phenotype. These data support an autosomal dominant mitochondrial uncoupling syndrome with hypermetabolism. (Funded by the National Institutes of Health.).
Asunto(s)
Enfermedades Mitocondriales , ATPasas de Translocación de Protón Mitocondriales , Fosforilación Oxidativa , Consumo de Oxígeno , Humanos , Masculino , Adenosina Trifosfato/metabolismo , Enfermedades en Gemelos/genética , Enfermedades en Gemelos/metabolismo , Fibroblastos/metabolismo , Mitocondrias/metabolismo , Enfermedades Mitocondriales/congénito , Enfermedades Mitocondriales/genética , Enfermedades Mitocondriales/metabolismo , ATPasas de Translocación de Protón Mitocondriales/genética , ATPasas de Translocación de Protón Mitocondriales/metabolismo , Mutación , Consumo de Oxígeno/genética , Consumo de Oxígeno/fisiología , Gemelos Monocigóticos/genéticaRESUMEN
The mammalian mitochondrial proteome is under dual genomic control, with 99% of proteins encoded by the nuclear genome and 13 originating from the mitochondrial DNA (mtDNA). We previously developed MitoCarta, a catalogue of over 1000 genes encoding the mammalian mitochondrial proteome. This catalogue was compiled using a Bayesian integration of multiple sequence features and experimental datasets, notably protein mass spectrometry of mitochondria isolated from fourteen murine tissues. Here, we introduce MitoCarta3.0. Beginning with the MitoCarta2.0 inventory, we performed manual review to remove 100 genes and introduce 78 additional genes, arriving at an updated inventory of 1136 human genes. We now include manually curated annotations of sub-mitochondrial localization (matrix, inner membrane, intermembrane space, outer membrane) as well as assignment to 149 hierarchical 'MitoPathways' spanning seven broad functional categories relevant to mitochondria. MitoCarta3.0, including sub-mitochondrial localization and MitoPathway annotations, is freely available at http://www.broadinstitute.org/mitocarta and should serve as a continued community resource for mitochondrial biology and medicine.
Asunto(s)
Bases de Datos de Proteínas , Mitocondrias/metabolismo , Proteínas Mitocondriales/metabolismo , Anotación de Secuencia Molecular , Proteoma/metabolismo , Animales , Teorema de Bayes , ADN Mitocondrial/genética , ADN Mitocondrial/metabolismo , Conjuntos de Datos como Asunto , Humanos , Internet , Aprendizaje Automático , Espectrometría de Masas , Ratones , Mitocondrias/genética , Membranas Mitocondriales/metabolismo , Proteínas Mitocondriales/clasificación , Proteínas Mitocondriales/genética , Proteoma/clasificación , Proteoma/genética , Programas InformáticosRESUMEN
The mitochondrial uniporter is a Ca2+-channel complex resident within the organelle's inner membrane. In mammalian cells the uniporter's activity is regulated by Ca2+ due to concerted action of MICU1 and MICU2, two paralogous, but functionally distinct, EF-hand Ca2+-binding proteins. Here we present the X-ray structure of the apo form of Mus musculus MICU2 at 2.5-Å resolution. The core structure of MICU2 is very similar to that of MICU1. It consists of two lobes, each containing one canonical Ca2+-binding EF-hand (EF1, EF4) and one structural EF-hand (EF2, EF3). Two molecules of MICU2 form a symmetrical dimer stabilized by highly conserved hydrophobic contacts between exposed residues of EF1 of one monomer and EF3 of another. Similar interactions stabilize MICU1 dimers, allowing exchange between homo- and heterodimers. The tight EF1-EF3 interface likely accounts for the structural and functional coupling between the Ca2+-binding sites in MICU1, MICU2, and their complex that leads to the previously reported Ca2+-binding cooperativity and dominant negative effect of mutation of the Ca2+-binding sites in either protein. The N- and C-terminal segments of the two proteins are distinctly different. In MICU2 the C-terminal helix is significantly longer than in MICU1, and it adopts a more rigid structure. MICU2's C-terminal helix is dispensable in vitro for its interaction with MICU1 but required for MICU2's function in cells. We propose that in the MICU1-MICU2 oligomeric complex the C-terminal helices of both proteins form a central semiautonomous assembly which contributes to the gating mechanism of the uniporter.
Asunto(s)
Canales de Calcio/química , Proteínas de Unión al Calcio/química , Proteínas de Transporte de Membrana Mitocondrial/química , Conformación Proteica , Animales , Sitios de Unión , Calcio/química , Canales de Calcio/genética , Proteínas de Unión al Calcio/genética , Cristalografía por Rayos X , Dimerización , Motivos EF Hand/genética , Células HeLa , Humanos , Ratones , Mitocondrias/química , Mitocondrias/genética , Proteínas de Transporte de Membrana Mitocondrial/genética , Estructura Secundaria de ProteínaRESUMEN
Hürthle cell carcinoma of the thyroid (HCC) is a form of thyroid cancer recalcitrant to radioiodine therapy that exhibits an accumulation of mitochondria. We performed whole-exome sequencing on a cohort of primary, recurrent, and metastatic tumors, and identified recurrent mutations in DAXX, TP53, NRAS, NF1, CDKN1A, ARHGAP35, and the TERT promoter. Parallel analysis of mtDNA revealed recurrent homoplasmic mutations in subunits of complex I of the electron transport chain. Analysis of DNA copy-number alterations uncovered widespread loss of chromosomes culminating in near-haploid chromosomal content in a large fraction of HCC, which was maintained during metastatic spread. This work uncovers a distinct molecular origin of HCC compared with other thyroid malignancies.
Asunto(s)
Aberraciones Cromosómicas , ADN Mitocondrial/genética , Mutación , Neoplasias de la Tiroides/genética , Variaciones en el Número de Copia de ADN , Haploidia , Humanos , Metástasis de la Neoplasia , Telomerasa/genética , Neoplasias de la Tiroides/patología , Secuenciación del ExomaRESUMEN
The mitochondrial uniporter is a Ca2+-activated Ca2+ channel complex that displays exceptionally high conductance and selectivity. Here, we report cellular metal toxicity screens highlighting the uniporter's role in Mn2+ toxicity. Cells lacking the pore-forming uniporter subunit, MCU, are more resistant to Mn2+ toxicity, while cells lacking the Ca2+-sensing inhibitory subunit, MICU1, are more sensitive than the wild type. Consistent with these findings, Caenorhabditis elegans lacking the uniporter's pore have increased resistance to Mn2+ toxicity. The chemical-genetic interaction between uniporter machinery and Mn2+ toxicity prompted us to hypothesize that Mn2+ can indeed be transported by the uniporter's pore, but this transport is prevented by MICU1. To this end, we demonstrate that, in the absence of MICU1, both Mn2+ and Ca2+ can pass through the uniporter, as evidenced by mitochondrial Mn2+ uptake assays, mitochondrial membrane potential measurements, and mitoplast electrophysiology. We show that Mn2+ does not elicit the conformational change in MICU1 that is physiologically elicited by Ca2+, preventing Mn2+ from inducing the pore opening. Our work showcases a mechanism by which a channel's auxiliary subunit can contribute to its apparent selectivity and, furthermore, may have implications for understanding how manganese contributes to neurodegenerative disease.
Asunto(s)
Canales de Calcio/metabolismo , Proteínas de Unión al Calcio/metabolismo , Calcio/metabolismo , Proteínas de Transporte de Catión/metabolismo , Manganeso/metabolismo , Proteínas de Transporte de Membrana Mitocondrial/metabolismo , Animales , Caenorhabditis elegans/genética , Caenorhabditis elegans/metabolismo , Proteínas de Caenorhabditis elegans/genética , Proteínas de Caenorhabditis elegans/metabolismo , Canales de Calcio/genética , Proteínas de Unión al Calcio/genética , Proteínas de Transporte de Catión/genética , Células HEK293 , Humanos , Transporte Iónico/fisiología , Células K562 , Proteínas de Transporte de Membrana Mitocondrial/genéticaRESUMEN
CLYBL encodes a ubiquitously expressed mitochondrial enzyme, conserved across all vertebrates, whose cellular activity and pathway assignment are unknown. Its homozygous loss is tolerated in seemingly healthy individuals, with reduced circulating B12 levels being the only and consistent phenotype reported to date. Here, by combining enzymology, structural biology, and activity-based metabolomics, we report that CLYBL operates as a citramalyl-CoA lyase in mammalian cells. Cells lacking CLYBL accumulate citramalyl-CoA, an intermediate in the C5-dicarboxylate metabolic pathway that includes itaconate, a recently identified human anti-microbial metabolite and immunomodulator. We report that CLYBL loss leads to a cell-autonomous defect in the mitochondrial B12 metabolism and that itaconyl-CoA is a cofactor-inactivating, substrate-analog inhibitor of the mitochondrial B12-dependent methylmalonyl-CoA mutase (MUT). Our work de-orphans the function of human CLYBL and reveals that a consequence of exposure to the immunomodulatory metabolite itaconate is B12 inactivation.
Asunto(s)
Liasas de Carbono-Carbono/metabolismo , Succinatos/metabolismo , Vitamina B 12/metabolismo , Liasas de Carbono-Carbono/química , Liasas de Carbono-Carbono/genética , Técnicas de Inactivación de Genes , Humanos , Redes y Vías Metabólicas , Mitocondrias/metabolismo , Modelos MolecularesRESUMEN
The redox coenzymes NADH and NADPH are broadly required for energy metabolism, biosynthesis and detoxification. Despite detailed knowledge of specific enzymes and pathways that utilize these coenzymes, a holistic understanding of the regulation and compartmentalization of NADH- and NADPH-dependent pathways is lacking, partly because of a lack of tools with which to investigate these processes in living cells. We have previously reported the use of the naturally occurring Lactobacillus brevis H2O-forming NADH oxidase (LbNOX) as a genetic tool for manipulation of the NAD+/NADH ratio in human cells. Here, we present triphosphopyridine nucleotide oxidase (TPNOX), a rationally designed and engineered mutant of LbNOX that is strictly specific to NADPH. We characterized the effects of TPNOX expression on cellular metabolism and used it in combination with LbNOX to show how the redox states of mitochondrial NADPH and NADH pools are connected.
Asunto(s)
NADH NADPH Oxidorreductasas/genética , NADH NADPH Oxidorreductasas/metabolismo , NADP/metabolismo , Ingeniería de Proteínas , Células HeLa , Humanos , NADH NADPH Oxidorreductasas/química , NADP/química , Oxidación-ReducciónRESUMEN
The mitochondrial calcium uniporter is a Ca2+-activated Ca2+ channel that is essential for dynamic modulation of mitochondrial function in response to cellular Ca2+ signals. It is regulated by two paralogous EF-hand proteins-MICU1 and MICU2, but the mechanism is unknown. Here, we demonstrate that both MICU1 and MICU2 are stabilized by Ca2+ We reconstitute the MICU1-MICU2 heterodimer and demonstrate that it binds Ca2+ cooperatively with high affinity. We discover that both MICU1 and MICU2 exhibit affinity for the mitochondria-specific lipid cardiolipin. We determine the minimum Ca2+ concentration required for disinhibition of the uniporter in permeabilized cells and report a close match with the Ca2+-binding affinity of MICU1-MICU2. We conclude that cooperative, high-affinity interaction of the MICU1-MICU2 complex with Ca2+ serves as an on-off switch, leading to a tightly controlled channel, capable of responding directly to cytosolic Ca2+ signals.
Asunto(s)
Canales de Calcio/metabolismo , Proteínas de Unión al Calcio/metabolismo , Calcio/metabolismo , Proteínas de Transporte de Catión/metabolismo , Proteínas de Transporte de Membrana Mitocondrial/metabolismo , Cardiolipinas/metabolismo , Citosol/metabolismo , Motivos EF Hand/fisiología , Células HEK293 , Células HeLa , Humanos , Potencial de la Membrana Mitocondrial , Mitocondrias/metabolismoRESUMEN
Mitochondria from many eukaryotic clades take up large amounts of calcium (Ca(2+)) via an inner membrane transporter called the uniporter. Transport by the uniporter is membrane potential dependent and sensitive to ruthenium red or its derivative Ru360 (ref. 1). Electrophysiological studies have shown that the uniporter is an ion channel with remarkably high conductance and selectivity. Ca(2+) entry into mitochondria is also known to activate the tricarboxylic acid cycle and seems to be crucial for matching the production of ATP in mitochondria with its cytosolic demand. Mitochondrial calcium uniporter (MCU) is the pore-forming and Ca(2+)-conducting subunit of the uniporter holocomplex, but its primary sequence does not resemble any calcium channel studied to date. Here we report the structure of the pore domain of MCU from Caenorhabditis elegans, determined using nuclear magnetic resonance (NMR) and electron microscopy (EM). MCU is a homo-oligomer in which the second transmembrane helix forms a hydrophilic pore across the membrane. The channel assembly represents a new solution of ion channel architecture, and is stabilized by a coiled-coil motif protruding into the mitochondrial matrix. The critical DXXE motif forms the pore entrance, which features two carboxylate rings; based on the ring dimensions and functional mutagenesis, these rings appear to form the selectivity filter. To our knowledge, this is one of the largest membrane protein structures characterized by NMR, and provides a structural blueprint for understanding the function of this channel.
Asunto(s)
Caenorhabditis elegans/química , Canales de Calcio/química , Secuencias de Aminoácidos , Animales , Canales de Calcio/genética , Canales de Calcio/metabolismo , Microscopía Electrónica , Mitocondrias/metabolismo , Modelos Moleculares , Resonancia Magnética Nuclear Biomolecular , Estructura Terciaria de Proteína , Relación Estructura-ActividadRESUMEN
A decline in electron transport chain (ETC) activity is associated with many human diseases. Although diminished mitochondrial adenosine triphosphate production is recognized as a source of pathology, the contribution of the associated reduction in the ratio of the amount of oxidized nicotinamide adenine dinucleotide (NAD(+)) to that of its reduced form (NADH) is less clear. We used a water-forming NADH oxidase from Lactobacillus brevis (LbNOX) as a genetic tool for inducing a compartment-specific increase of the NAD(+)/NADH ratio in human cells. We used LbNOX to demonstrate the dependence of key metabolic fluxes, gluconeogenesis, and signaling on the cytosolic or mitochondrial NAD(+)/NADH ratios. Expression of LbNOX in the cytosol or mitochondria ameliorated proliferative and metabolic defects caused by an impaired ETC. The results underscore the role of reductive stress in mitochondrial pathogenesis and demonstrate the utility of targeted LbNOX for direct, compartment-specific manipulation of redox state.
Asunto(s)
Proteínas del Complejo de Cadena de Transporte de Electrón/metabolismo , Gluconeogénesis/genética , Mitocondrias/metabolismo , Complejos Multienzimáticos/metabolismo , NADH NADPH Oxidorreductasas/metabolismo , NAD/metabolismo , Catálisis , Citosol/enzimología , Transporte de Electrón , Proteínas del Complejo de Cadena de Transporte de Electrón/genética , Prueba de Complementación Genética , Células HeLa , Humanos , Levilactobacillus brevis/enzimología , Levilactobacillus brevis/genética , Enfermedades Mitocondriales/enzimología , Enfermedades Mitocondriales/genética , Complejos Multienzimáticos/genética , NADH NADPH Oxidorreductasas/genética , Oxidación-ReducciónRESUMEN
CLYBL is a human mitochondrial enzyme of unknown function that is found in multiple eukaryotic taxa and conserved to bacteria. The protein is expressed in the mitochondria of all mammalian organs, with highest expression in brown fat and kidney. Approximately 5% of all humans harbor a premature stop polymorphism in CLYBL that has been associated with reduced levels of circulating vitamin B12. Using comparative genomics, we now show that CLYBL is strongly co-expressed with and co-evolved specifically with other components of the mitochondrial B12 pathway. We confirm that the premature stop polymorphism in CLYBL leads to a loss of protein expression. To elucidate the molecular function of CLYBL, we used comparative operon analysis, structural modeling and enzyme kinetics. We report that CLYBL encodes a malate/ß-methylmalate synthase, converting glyoxylate and acetyl-CoA to malate, or glyoxylate and propionyl-CoA to ß-methylmalate. Malate synthases are best known for their established role in the glyoxylate shunt of plants and lower organisms and are traditionally described as not occurring in humans. The broader role of a malate/ß-methylmalate synthase in human physiology and its mechanistic link to vitamin B12 metabolism remain unknown.
Asunto(s)
Enzimas/metabolismo , Malato Sintasa/metabolismo , Oxo-Ácido-Liasas/metabolismo , Acetilcoenzima A/metabolismo , Acilcoenzima A/metabolismo , Glioxilatos/metabolismo , Humanos , Malatos/metabolismo , Especificidad por SustratoRESUMEN
Dr. John Gergely passed away on July 26, 2013 after a long and distinguished career. His publications spanned 67 years. He founded the Department of Muscle Research in the Retina Foundation (which later became the Boston Biomedical Research Institute) and served as director for 34 years. Dr. Gergely served on the editorial boards of ten scientific journals. He was elected as a Fellow of both the Biophysical Society and the American Association for the Advancement of Science. Dr. Gergely made major contributions concerning muscle protein structure and function. He was best known for his work on the troponin complex. The insights of John and his associates have provided the foundation for our understanding of calcium regulation in skeletal and cardiac muscle.
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Bioquímica/historia , Fisiología/historia , Investigación Biomédica , Historia del Siglo XX , Historia del Siglo XXI , Humanos , Proteínas Musculares/químicaRESUMEN
Cardiac muscle contraction is activated via the single Ca(2+)-binding site (site II) in the N-domain of troponin C (cTnC). The two Ca(2+)/Mg(2+) binding sites in the C-domain of cTnC (sites III and IV) have been considered to play a purely structural role in anchoring cTnC to the thin filament. However, several recent discoveries suggest a possible role of this domain in contractile regulation. The green tea polyphenol (-)-epigallocatechin 3-gallate (EGCg), which binds specifically to the C-domain of cTnC, reduces cardiac myofilament Ca(2+) sensitivity along with maximum force and acto-myosin ATPase activity. We have determined the effect of EGCg on Ca(2+) and Mg(2+) binding to the C-domain of cTnC. In the absence of Mg(2+) there was no significant effect of EGCg on the Ca(2+)-cTnC affinity. Surprisingly, in the presence of Mg(2+) EGCg caused an increase in Ca(2+) affinity for sites III and IV of cTnC. However, in the absence of Ca(2+) the addition of EGCg caused a significant reduction in Mg(2+)-cTnC affinity. This reduction is presumably responsible for the increase in Ca(2+)-cTnC affinity produced by EGCg in the presence of Mg(2+). We propose that the inhibitory effect of EGCg on myofilament Ca(2+) activation may be related to an enhanced Ca(2+)-Mg(2+)exchange at sites III and IV of cTnC, which might reduce the myosin crossbridge dependent component of thin filament activation.
Asunto(s)
Anticarcinógenos/farmacología , Catequina/análogos & derivados , Magnesio/metabolismo , Contracción Miocárdica/efectos de los fármacos , Miocardio/metabolismo , Troponina C/metabolismo , Actomiosina/genética , Actomiosina/metabolismo , Animales , Anticarcinógenos/química , Calcio/metabolismo , Señalización del Calcio/efectos de los fármacos , Catequina/química , Catequina/farmacología , Pollos/genética , Pollos/metabolismo , Electrocardiografía , Unión Proteica/efectos de los fármacos , Estructura Terciaria de Proteína , Ratas , Té/química , Troponina C/genéticaRESUMEN
CaMKII (Ca²âº/calmodulin-dependent kinase II) is a serine/threonine phosphotransferase that is capable of long-term retention of activity due to autophosphorylation at a specific threonine residue within each subunit of its oligomeric structure. The γ isoform of CaMKII is a significant regulator of vascular contractility. Here, we show that phosphorylation of CaMKII γ at Ser²6, a residue located within the ATP-binding site, terminates the sustained activity of the enzyme. To test the physiological importance of phosphorylation at Ser²6, we generated a phosphospecific Ser²6 antibody and demonstrated an increase in Ser²6 phosphorylation upon depolarization and contraction of blood vessels. To determine if the phosphorylation of Ser²6 affects the kinase activity, we mutated Ser²6 to alanine or aspartic acid. The S26D mutation mimicking the phosphorylated state of CaMKII causes a dramatic decrease in Thr²87 autophosphorylation levels and greatly reduces the catalytic activity towards an exogenous substrate (autocamtide-3), whereas the S26A mutation has no effect. These data combined with molecular modelling indicate that a negative charge at Ser²6 of CaMKII γ inhibits the catalytic activity of the enzyme towards its autophosphorylation site at Thr²87 most probably by blocking ATP binding. We propose that Ser²6 phosphorylation constitutes an important mechanism for switching off CaMKII activity.
Asunto(s)
Adenosina Trifosfato/metabolismo , Proteína Quinasa Tipo 2 Dependiente de Calcio Calmodulina/metabolismo , Calcio/metabolismo , Isoformas de Proteínas/metabolismo , Animales , Sitios de Unión , Proteína Quinasa Tipo 2 Dependiente de Calcio Calmodulina/genética , Calmodulina/genética , Calmodulina/metabolismo , Hurones/genética , Hurones/metabolismo , Mutación , Fosforilación/genética , Isoformas de Proteínas/genética , Serina/genética , Serina/metabolismo , Transducción de Señal , Treonina/genética , Treonina/metabolismoRESUMEN
Calmodulin (CaM), a member of the EF-hand superfamily, regulates many aspects of cell function by responding specifically to micromolar concentrations of Ca(2+) in the presence of an ~1000-fold higher concentration of cellular Mg(2+). To explain the structural basis of metal ion binding specificity, we have determined the X-ray structures of the N-terminal domain of calmodulin (N-CaM) in complexes with Mg(2+), Mn(2+), and Zn(2+). In contrast to Ca(2+), which induces domain opening in CaM, octahedrally coordinated Mg(2+) and Mn(2+) stabilize the closed-domain, apo-like conformation, while tetrahedrally coordinated Zn(2+) ions bind at the protein surface and do not compete with Ca(2+). The relative positions of bound Mg(2+) and Mn(2+) within the EF-hand loops are similar to those of Ca(2+); however, the Glu side chain at position 12 of the loop, whose bidentate interaction with Ca(2+) is critical for domain opening, does not bind directly to either Mn(2+) or Mg(2+), and the vacant ligand position is occupied by a water molecule. We conclude that this critical interaction is prevented by specific stereochemical constraints imposed on the ligands by the EF-hand ß-scaffold. The structures suggest that Mg(2+) contributes to the switching off of calmodulin activity and possibly other EF-hand proteins at the resting levels of Ca(2+). The Mg(2+)-bound N-CaM structure also provides a unique view of a transiently bound hydrated metal ion and suggests a role for the hydration water in the metal-induced conformational change.
Asunto(s)
Calmodulina/química , Calmodulina/metabolismo , Motivos EF Hand , Magnesio/metabolismo , Manganeso/metabolismo , Cristalografía por Rayos X , Humanos , Modelos Moleculares , Unión Proteica , Estabilidad Proteica , Estructura Terciaria de Proteína , Especificidad por Sustrato , Zinc/metabolismoRESUMEN
The Ca(2+)/Mg(2+) sites (III and IV) located in the C-terminal domain of cardiac troponin C (cTnC) have been generally considered to play a purely structural role in keeping the cTnC bound to the thin filament. However, several lines of evidence, including the discovery of cardiomyopathy-associated mutations in the C-domain, have raised the possibility that these sites may have a more complex role in contractile regulation. To explore this possibility, the ATPase activity of rat cardiac myofibrils was assayed under conditions in which no Ca(2+) was bound to the N-terminal regulatory Ca(2+)-binding site (site II). Myosin-S1 was treated with N-ethylmaleimide to create strong-binding myosin heads (NEM-S1), which could activate the cardiac thin filament in the absence of Ca(2+). NEM-S1 activation was assayed at pCa 8.0 to 6.5 and in the presence of either 1mM or 30 µM free Mg(2+). ATPase activity was maximal when sites III and IV were occupied by Mg(2+) and it steadily declined as Ca(2+) displaced Mg(2+). The data suggest that in the absence of Ca(2+) at site II strong-binding myosin crossbridges cause the opening of more active sites on the thin filament if the C-domain is occupied by Mg(2+) rather than Ca(2+). This finding could be relevant to the contraction-relaxation kinetics of cardiac muscle. As Ca(2+) dissociates from site II of cTnC during the early relaxing phase of the cardiac cycle, residual Ca(2+) bound at sites III and IV might facilitate the switching off of the thin filament and the detachment of crossbridges from actin.
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Citoesqueleto de Actina/metabolismo , Calcio/metabolismo , Magnesio/metabolismo , Miocardio/metabolismo , Miofibrillas/metabolismo , Troponina C/metabolismo , Adenosina Trifosfatasas/metabolismo , Animales , Contracción Miocárdica , RatasRESUMEN
The Ca(2+)-binding helix-loop-helix structural motif called "EF-hand" is a common building block of a large family of proteins that function as intracellular Ca(2+)-receptors. These proteins respond specifically to micromolar concentrations of Ca(2+) in the presence of ~1000-fold excess of the chemically similar divalent cation Mg(2+). The intracellular free Mg(2+) concentration is tightly controlled in a narrow range of 0.5-1.0mM, which at the resting Ca(2+) levels is sufficient to fully or partially saturate the Ca(2+)-binding sites of many EF-hand proteins. Thus, to convey Ca(2+) signals, EF-hand proteins must respond differently to Ca(2+) than to Mg(2+). In this review the structural aspects of Mg(2+) binding to EF-hand proteins are considered and interpreted in light of the recently proposed two-step Ca(2+)-binding mechanism (Grabarek, Z., J. Mol. Biol., 2005, 346, 1351). It is proposed that, due to stereochemical constraints imposed by the two-EF-hand domain structure, the smaller Mg(2+) ion cannot engage the ligands of an EF-hand in the same way as Ca(2+) and defaults to stabilizing the apo-like conformation of the EF-hand. It is proposed that Mg(2+) plays an active role in the Ca(2+)-dependent regulation of cellular processes by stabilizing the "off state" of some EF-hand proteins, thereby facilitating switching off their respective target enzymes at the resting Ca(2+) levels. Therefore, some pathological conditions attributed to Mg(2+) deficiency might be related to excessive activation of underlying Ca(2+)-regulated cellular processes. This article is part of a Special Issue entitled: 11th European Symposium on Calcium.
Asunto(s)
Señalización del Calcio , Calmodulina/metabolismo , Motivos EF Hand , Magnesio/metabolismo , Troponina C/metabolismo , Animales , Humanos , Magnesio/química , Modelos MolecularesRESUMEN
Smooth muscle myosin light chain kinase (smMLCK) is a calcium-calmodulin complex-dependent enzyme that activates contraction of smooth muscle. The polypeptide chain of rabbit uterine smMLCK (Swiss-Prot entry P29294) contains the catalytic/regulatory domain, three immunoglobulin-related motifs (Ig), one fibronectin-related motif (Fn3), a repetitive, proline-rich segment (PEVK), and, at the N-terminus, a unique F-actin-binding domain. We have evaluated the spatial arrangement of these domains in a recombinant 125 kDa full-length smMLCK and its two catalytically active C-terminal fragments (77 kDa, residues 461-1147, and 61 kDa, residues 461-1002). Electron microscopic images of smMLCK cross-linked to F-actin show particles at variable distances (11-55 nm) from the filament, suggesting that a well-structured C-terminal segment of smMLCK is connected to the actin-binding domain by a long, flexible tether. We have used structural homology and molecular dynamics methods to construct various all-atom representation models of smMLCK and its two fragments. The theoretical sedimentation coefficients computed with HYDROPRO were compared with those determined by sedimentation velocity. We found agreement between the predicted and observed sedimentation coefficients for models in which the independently folded catalytic domain, Fn3, and Ig domains are aligned consecutively on the long axis of the molecule. The PEVK segment is modeled as an extensible linker that enables smMLCK to remain bound to F-actin and simultaneously activate the myosin heads of adjacent myosin filaments at a distance of >or=40 nm. The structural properties of smMLCK may contribute to the elasticity of smooth muscle cells.
Asunto(s)
Músculo Liso/enzimología , Quinasa de Cadena Ligera de Miosina/química , Actinas/química , Actinas/metabolismo , Animales , Sitios de Unión , Elasticidad , Femenino , Microscopía Electrónica , Quinasa de Cadena Ligera de Miosina/metabolismo , Unión Proteica , Conformación Proteica , Conejos , ÚteroRESUMEN
The actin-binding protein caldesmon (CaD) reversibly inhibits smooth muscle contraction. In non-muscle cells, a shorter CaD isoform co-exists with microfilaments in the stress fibers at the quiescent state, but the phosphorylated CaD is found at the leading edge of migrating cells where dynamic actin filament remodeling occurs. We have studied the effect of a C-terminal fragment of CaD (H32K) on the kinetics of the in vitro actin polymerization by monitoring the fluorescence of pyrene-labeled actin. Addition of H32K or its phosphorylated form either attenuated or accelerated the pyrene emission enhancement, depending on whether it was added at the early or the late phase of actin polymerization. However, the CaD fragment had no effect on the yield of sedimentable actin, nor did it affect the actin ATPase activity. Our findings can be explained by a model in which nascent actin filaments undergo a maturation process that involves at least two intermediate conformational states. If present at early stages of actin polymerization, CaD stabilizes one of the intermediate states and blocks the subsequent filament maturation. Addition of CaD at a later phase accelerates F-actin formation. The fact that CaD is capable of inhibiting actin filament maturation provides a novel function for CaD and suggests an active role in the dynamic reorganization of the actin cytoskeleton.