RESUMEN
Mitochondrial energy conversion requires an intricate architecture of the inner mitochondrial membrane1. Here we show that a supercomplex containing all four respiratory chain components contributes to membrane curvature induction in ciliates. We report cryo-electron microscopy and cryo-tomography structures of the supercomplex that comprises 150 different proteins and 311 bound lipids, forming a stable 5.8-MDa assembly. Owing to subunit acquisition and extension, complex I associates with a complex IV dimer, generating a wedge-shaped gap that serves as a binding site for complex II. Together with a tilted complex III dimer association, it results in a curved membrane region. Using molecular dynamics simulations, we demonstrate that the divergent supercomplex actively contributes to the membrane curvature induction and tubulation of cristae. Our findings highlight how the evolution of protein subunits of respiratory complexes has led to the I-II-III2-IV2 supercomplex that contributes to the shaping of the bioenergetic membrane, thereby enabling its functional specialization.
Asunto(s)
Microscopía por Crioelectrón , Complejo III de Transporte de Electrones , Complejo II de Transporte de Electrones , Complejo IV de Transporte de Electrones , Complejo I de Transporte de Electrón , Mitocondrias , Membranas Mitocondriales , Transporte de Electrón , Complejo III de Transporte de Electrones/química , Complejo III de Transporte de Electrones/metabolismo , Complejo III de Transporte de Electrones/ultraestructura , Complejo IV de Transporte de Electrones/química , Complejo IV de Transporte de Electrones/metabolismo , Complejo IV de Transporte de Electrones/ultraestructura , Mitocondrias/química , Mitocondrias/enzimología , Mitocondrias/metabolismo , Mitocondrias/ultraestructura , Membranas Mitocondriales/química , Membranas Mitocondriales/enzimología , Membranas Mitocondriales/metabolismo , Membranas Mitocondriales/ultraestructura , Complejo II de Transporte de Electrones/química , Complejo II de Transporte de Electrones/metabolismo , Complejo II de Transporte de Electrones/ultraestructura , Complejo I de Transporte de Electrón/química , Complejo I de Transporte de Electrón/metabolismo , Complejo I de Transporte de Electrón/ultraestructura , Multimerización de Proteína , Subunidades de Proteína/química , Subunidades de Proteína/metabolismo , Simulación de Dinámica Molecular , Sitios de Unión , Evolución MolecularRESUMEN
AMPA glutamate receptors (AMPARs), the primary mediators of excitatory neurotransmission in the brain, are either GluA2 subunit-containing and thus Ca2+-impermeable, or GluA2-lacking and Ca2+-permeable1. Despite their prominent expression throughout interneurons and glia, their role in long-term potentiation and their involvement in a range of neuropathologies2, structural information for GluA2-lacking receptors is currently absent. Here we determine and characterize cryo-electron microscopy structures of the GluA1 homotetramer, fully occupied with TARPγ3 auxiliary subunits (GluA1/γ3). The gating core of both resting and open-state GluA1/γ3 closely resembles GluA2-containing receptors. However, the sequence-diverse N-terminal domains (NTDs) give rise to a highly mobile assembly, enabling domain swapping and subunit re-alignments in the ligand-binding domain tier that are pronounced in desensitized states. These transitions underlie the unique kinetic properties of GluA1. A GluA2 mutant (F231A) increasing NTD dynamics phenocopies this behaviour, and exhibits reduced synaptic responses, reflecting the anchoring function of the AMPAR NTD at the synapse. Together, this work underscores how the subunit-diverse NTDs determine subunit arrangement, gating properties and ultimately synaptic signalling efficiency among AMPAR subtypes.
Asunto(s)
Ácido Glutámico , Transmisión Sináptica , Ácido alfa-Amino-3-hidroxi-5-metil-4-isoxazol Propiónico , Microscopía por Crioelectrón , Sinapsis/fisiologíaRESUMEN
The autosomal dominant monogenetic disease neurofibromatosis type 1 (NF1) affects approximately one in 3,000 individuals and is caused by mutations in the NF1 tumour suppressor gene, leading to dysfunction in the protein neurofibromin (Nf1)1,2. As a GTPase-activating protein, a key function of Nf1 is repression of the Ras oncogene signalling cascade. We determined the human Nf1 dimer structure at an overall resolution of 3.3 Å. The cryo-electron microscopy structure reveals domain organization and structural details of the Nf1 exon 23a splicing3 isoform 2 in a closed, self-inhibited, Zn-stabilized state and an open state. In the closed conformation, HEAT/ARM core domains shield the GTPase-activating protein-related domain (GRD) so that Ras binding is sterically inhibited. In a distinctly different, open conformation of one protomer, a large-scale movement of the GRD occurs, which is necessary to access Ras, whereas Sec14-PH reorients to allow interaction with the cellular membrane4. Zn incubation of Nf1 leads to reduced Ras-GAP activity with both protomers in the self-inhibited, closed conformation stabilized by a Zn binding site between the N-HEAT/ARM domain and the GRD-Sec14-PH linker. The transition between closed, self-inhibited states of Nf1 and open states provides guidance for targeted studies deciphering the complex molecular mechanism behind the widespread neurofibromatosis syndrome and Nf1 dysfunction in carcinogenesis.
Asunto(s)
Microscopía por Crioelectrón , Neurofibromina 2/química , Neurofibromina 2/metabolismo , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Empalme Alternativo , Sitios de Unión , Exones , Humanos , Modelos Moleculares , Neurofibromina 1/metabolismo , Neurofibromina 2/ultraestructura , Unión Proteica , Dominios Proteicos , Isoformas de Proteínas/química , Isoformas de Proteínas/metabolismo , Isoformas de Proteínas/ultraestructura , Multimerización de Proteína , Estabilidad Proteica , Zinc/metabolismoRESUMEN
Mitoribosomes consist of ribosomal RNA and protein components, coordinated assembly of which is critical for function. We used mitoribosomes from Trypanosoma brucei with reduced RNA and increased protein mass to provide insights into the biogenesis of the mitoribosomal large subunit. Structural characterization of a stable assembly intermediate revealed 22 assembly factors, some of which have orthologues/counterparts/homologues in mammalian genomes. These assembly factors form a protein network that spans a distance of 180 Å, shielding the ribosomal RNA surface. The central protuberance and L7/L12 stalk are not assembled entirely and require removal of assembly factors and remodeling of the mitoribosomal proteins to become functional. The conserved proteins GTPBP7 and mt-EngA are bound together at the subunit interface in proximity to the peptidyl transferase center. A mitochondrial acyl-carrier protein plays a role in docking the L1 stalk, which needs to be repositioned during maturation. Additional enzymatically deactivated factors scaffold the assembly while the exit tunnel is blocked. Together, this extensive network of accessory factors stabilizes the immature sites and connects the functionally important regions of the mitoribosomal large subunit.
Asunto(s)
Proteínas de Unión al GTP/metabolismo , Ribosomas Mitocondriales/metabolismo , Biosíntesis de Proteínas/fisiología , Subunidades Ribosómicas Grandes/metabolismo , Trypanosoma brucei brucei/metabolismo , Microscopía por Crioelectrón , Unión Proteica/fisiología , Conformación Proteica , ARN Ribosómico/genéticaRESUMEN
The mitochondrial calcium uniporter (MCU) is a highly selective calcium channel and a major route of calcium entry into mitochondria. How the channel catalyses ion permeation and achieves ion selectivity are not well understood, partly because MCU is thought to have a distinct architecture in comparison to other cellular channels. Here we report cryo-electron microscopy reconstructions of MCU channels from zebrafish and Cyphellophora europaea at 8.5 Å and 3.2 Å resolutions, respectively. In contrast to a previous report of pentameric stoichiometry for MCU, both channels are tetramers. The atomic model of C. europaea MCU shows that a conserved WDXXEP signature sequence forms the selectivity filter, in which calcium ions are arranged in single file. Coiled-coil legs connect the pore to N-terminal domains in the mitochondrial matrix. In C. europaea MCU, the N-terminal domains assemble as a dimer of dimers; in zebrafish MCU, they form an asymmetric crescent. The structures define principles that underlie ion permeation and calcium selectivity in this unusual channel.
Asunto(s)
Canales de Calcio/química , Canales de Calcio/ultraestructura , Microscopía por Crioelectrón , Phialophora/química , Pez Cebra , Animales , Caenorhabditis elegans/química , Calcio/metabolismo , Canales de Calcio/metabolismo , Activación del Canal Iónico , Modelos Moleculares , Multimerización de Proteína , Subunidades de Proteína/química , Subunidades de Proteína/metabolismoRESUMEN
Complex I is the first and largest enzyme of the respiratory chain and has a central role in cellular energy production through the coupling of NADH:ubiquinone electron transfer to proton translocation. It is also implicated in many common human neurodegenerative diseases. Here, we report the first crystal structure of the entire, intact complex I (from Thermus thermophilus) at 3.3 Å resolution. The structure of the 536-kDa complex comprises 16 different subunits, with a total of 64 transmembrane helices and 9 iron-sulphur clusters. The core fold of subunit Nqo8 (ND1 in humans) is, unexpectedly, similar to a half-channel of the antiporter-like subunits. Small subunits nearby form a linked second half-channel, which completes the fourth proton-translocation pathway (present in addition to the channels in three antiporter-like subunits). The quinone-binding site is unusually long, narrow and enclosed. The quinone headgroup binds at the deep end of this chamber, near iron-sulphur cluster N2. Notably, the chamber is linked to the fourth channel by a 'funnel' of charged residues. The link continues over the entire membrane domain as a flexible central axis of charged and polar residues, and probably has a leading role in the propagation of conformational changes, aided by coupling elements. The structure suggests that a unique, out-of-the-membrane quinone-reaction chamber enables the redox energy to drive concerted long-range conformational changes in the four antiporter-like domains, resulting in translocation of four protons per cycle.
Asunto(s)
Complejo I de Transporte de Electrón/química , Complejo I de Transporte de Electrón/metabolismo , Thermus thermophilus/química , Benzoquinonas/química , Benzoquinonas/metabolismo , Membrana Celular/metabolismo , Cristalografía por Rayos X , Humanos , Interacciones Hidrofóbicas e Hidrofílicas , Modelos Moleculares , NAD/metabolismo , Oxidación-Reducción , Pliegue de Proteína , Subunidades de Proteína/química , Subunidades de Proteína/metabolismo , Fuerza Protón-Motriz , Protones , Thermus thermophilus/citología , Ubiquinona/metabolismoRESUMEN
Complex I (NADH:ubiquinone oxidoreductase) plays a central role in cellular energy production, coupling electron transfer between NADH and quinone to proton translocation. It is the largest protein assembly of respiratory chains and one of the most elaborate redox membrane proteins known. Bacterial enzyme is about half the size of mitochondrial and thus provides its important "minimal" model. Dysfunction of mitochondrial complex I is implicated in many human neurodegenerative diseases. The L-shaped complex consists of a hydrophilic arm, where electron transfer occurs, and a membrane arm, where proton translocation takes place. We have solved the crystal structures of the hydrophilic domain of complex I from Thermus thermophilus, the membrane domain from Escherichia coli and recently of the intact, entire complex I from T. thermophilus (536 kDa, 16 subunits, 9 iron-sulphur clusters, 64 transmembrane helices). The 95Å long electron transfer pathway through the enzyme proceeds from the primary electron acceptor flavin mononucleotide through seven conserved Fe-S clusters to the unusual elongated quinone-binding site at the interface with the membrane domain. Four putative proton translocation channels are found in the membrane domain, all linked by the central flexible axis containing charged residues. The redox energy of electron transfer is coupled to proton translocation by the as yet undefined mechanism proposed to involve long-range conformational changes. This article is part of a Special Issue entitled Respiratory complex I, edited by Volker Zickermann and Ulrich Brandt.
Asunto(s)
Proteínas Bacterianas/química , Proteínas Bacterianas/ultraestructura , Complejo I de Transporte de Electrón/química , Complejo I de Transporte de Electrón/ultraestructura , Modelos Químicos , Simulación de Dinámica Molecular , Transporte de Electrón , Conformación Proteica , Bombas de Protones/química , Bombas de Protones/ultraestructura , Relación Estructura-ActividadRESUMEN
Complex I is the first enzyme of the respiratory chain and has a central role in cellular energy production, coupling electron transfer between NADH and quinone to proton translocation by an unknown mechanism. Dysfunction of complex I has been implicated in many human neurodegenerative diseases. We have determined the structure of its hydrophilic domain previously. Here, we report the alpha-helical structure of the membrane domain of complex I from Escherichia coli at 3.9 A resolution. The antiporter-like subunits NuoL/M/N each contain 14 conserved transmembrane (TM) helices. Two of them are discontinuous, as in some transporters. Unexpectedly, subunit NuoL also contains a 110-A long amphipathic alpha-helix, spanning almost the entire length of the domain. Furthermore, we have determined the structure of the entire complex I from Thermus thermophilus at 4.5 A resolution. The L-shaped assembly consists of the alpha-helical model for the membrane domain, with 63 TM helices, and the known structure of the hydrophilic domain. The architecture of the complex provides strong clues about the coupling mechanism: the conformational changes at the interface of the two main domains may drive the long amphipathic alpha-helix of NuoL in a piston-like motion, tilting nearby discontinuous TM helices, resulting in proton translocation.
Asunto(s)
Complejo I de Transporte de Electrón/química , Complejo I de Transporte de Electrón/metabolismo , Escherichia coli/enzimología , Subunidades de Proteína/química , Subunidades de Proteína/metabolismo , Thermus thermophilus/enzimología , Benzoquinonas/metabolismo , Sitios de Unión , Membrana Celular/metabolismo , Cristalografía por Rayos X , Modelos Moleculares , Estructura Secundaria de Proteína , Estructura Terciaria de Proteína , Relación Estructura-ActividadRESUMEN
Complex I (NADH:ubiquinone oxidoreductase) is central to cellular energy production, being the first and largest enzyme of the respiratory chain in mitochondria. It couples electron transfer from NADH to ubiquinone with proton translocation across the inner mitochondrial membrane and is involved in a wide range of human neurodegenerative disorders. Mammalian complex I is composed of 44 different subunits, whereas the 'minimal' bacterial version contains 14 highly conserved 'core' subunits. The L-shaped assembly consists of hydrophilic and membrane domains. We have determined all known atomic structures of complex I, starting from the hydrophilic domain of Thermus thermophilus enzyme (eight subunits, nine Fe-S clusters), followed by the membrane domains of the Escherichia coli (six subunits, 55 transmembrane helices) and T. thermophilus (seven subunits, 64 transmembrane helices) enzymes, and finally culminating in a recent crystal structure of the entire intact complex I from T. thermophilus (536 kDa, 16 subunits, nine Fe-S clusters, 64 transmembrane helices). The structure suggests an unusual and unique coupling mechanism via long-range conformational changes. Determination of the structure of the entire complex was possible only through this step-by-step approach, building on from smaller subcomplexes towards the entire assembly. Large membrane proteins are notoriously difficult to crystallize, and so various non-standard and sometimes counterintuitive approaches were employed in order to achieve crystal diffraction to high resolution and solve the structures. These steps, as well as the implications from the final structure, are discussed in the present review.
Asunto(s)
Complejo I de Transporte de Electrón/química , Complejos Multiproteicos/química , Conformación Proteica , Bombas de Protones/química , Metabolismo Energético , Escherichia coli/enzimología , Humanos , Membranas Mitocondriales/química , Modelos Moleculares , Estructura Terciaria de Proteína , Subunidades de Proteína/química , Thermus thermophilus/enzimologíaRESUMEN
The proteins MCU and EMRE form the minimal functional unit of the mitochondrial calcium uniporter complex in metazoans, a highly selective and tightly controlled Ca2+ channel of the inner mitochondrial membrane that regulates cellular metabolism. Here we present functional reconstitution of an MCU-EMRE complex from the red flour beetle, Tribolium castaneum, and a cryo-EM structure of the complex at 3.5â¯Å resolution. Using a novel assay, we demonstrate robust Ca2+ uptake into proteoliposomes containing the purified complex. Uptake is dependent on EMRE and also on the mitochondrial lipid cardiolipin. The structure reveals a tetrameric channel with a single ion pore. EMRE is located at the periphery of the transmembrane domain and associates primarily with the first transmembrane helix of MCU. Coiled-coil and juxtamembrane domains within the matrix portion of the complex adopt markedly different conformations than in a structure of a human MCU-EMRE complex, suggesting that the structures represent different conformations of these functionally similar metazoan channels.
Asunto(s)
Canales de Calcio/química , Mitocondrias/metabolismo , Proteínas de Transporte de Membrana Mitocondrial/química , Membranas Mitocondriales/metabolismo , Animales , Calcio/metabolismo , Proteínas de Unión al Calcio/química , Proteínas de Transporte de Catión/química , Humanos , Proteínas de Transporte de Membrana Mitocondrial/metabolismo , Modelos Moleculares , Conformación Proteica , Dominios ProteicosRESUMEN
The mitochondrial calcium uniporter is a Ca2+-gated ion channel complex that controls mitochondrial Ca2+ entry and regulates cell metabolism. MCU and EMRE form the channel while Ca2+-dependent regulation is conferred by MICU1 and MICU2 through an enigmatic process. We present a cryo-EM structure of an MCU-EMRE-MICU1-MICU2 holocomplex comprising MCU and EMRE subunits from the beetle Tribolium castaneum in complex with a human MICU1-MICU2 heterodimer at 3.3 Å resolution. With analogy to how neuronal channels are blocked by protein toxins, a uniporter interaction domain on MICU1 binds to a channel receptor site comprising MCU and EMRE subunits to inhibit ion flow under resting Ca2+ conditions. A Ca2+-bound structure of MICU1-MICU2 at 3.1 Å resolution indicates how Ca2+-dependent changes enable dynamic response to cytosolic Ca2+ signals.
Asunto(s)
Canales de Calcio/metabolismo , Proteínas de Unión al Calcio/metabolismo , Proteínas de Transporte de Catión/metabolismo , Proteínas de Insectos/metabolismo , Proteínas de Transporte de Membrana Mitocondrial/metabolismo , Tribolium/genética , Animales , Microscopía por Crioelectrón , Proteínas de Drosophila , Genes de Insecto , Células HEK293 , Humanos , Tribolium/metabolismoRESUMEN
Complex I is the first and the largest enzyme of respiratory chains in bacteria and mitochondria. The mechanism which couples spatially separated transfer of electrons to proton translocation in complex I is not known. Here we report five crystal structures of T. thermophilus enzyme in complex with NADH or quinone-like compounds. We also determined cryo-EM structures of major and minor native states of the complex, differing in the position of the peripheral arm. Crystal structures show that binding of quinone-like compounds (but not of NADH) leads to a related global conformational change, accompanied by local re-arrangements propagating from the quinone site to the nearest proton channel. Normal mode and molecular dynamics analyses indicate that these are likely to represent the first steps in the proton translocation mechanism. Our results suggest that quinone binding and chemistry play a key role in the coupling mechanism of complex I.