RESUMEN
Cellular respiration is powered by membrane-bound redox enzymes that convert chemical energy into an electrochemical proton gradient and drive the energy metabolism. By combining large-scale classical and quantum mechanical simulations with cryo-electron microscopy data, we resolve here molecular details of conformational changes linked to proton pumping in the mammalian complex I. Our data suggest that complex I deactivation blocks water-mediated proton transfer between a membrane-bound quinone site and proton-pumping modules, decoupling the energy-transduction machinery. We identify a putative gating region at the interface between membrane domain subunits ND1 and ND3/ND4L/ND6 that modulates the proton transfer by conformational changes in transmembrane helices and bulky residues. The region is perturbed by mutations linked to human mitochondrial disorders and is suggested to also undergo conformational changes during catalysis of simpler complex I variants that lack the "active"-to-"deactive" transition. Our findings suggest that conformational changes in transmembrane helices modulate the proton transfer dynamics by wetting/dewetting transitions and provide important functional insight into the mammalian respiratory complex I.
Asunto(s)
Complejo I de Transporte de Electrón/química , Complejo I de Transporte de Electrón/metabolismo , Protones , Animales , Sitios de Unión , Transporte Biológico , Respiración de la Célula , Microscopía por Crioelectrón , Complejo I de Transporte de Electrón/genética , Metabolismo Energético , Humanos , Enfermedades Mitocondriales/genética , Membranas Mitocondriales/química , Membranas Mitocondriales/metabolismo , Simulación de Dinámica Molecular , Mutación , Oxidación-Reducción , Conformación Proteica , Dominios Proteicos , Estructura Secundaria de Proteína , Quinonas/química , Quinonas/metabolismo , Agua/química , Agua/metabolismoRESUMEN
The heat shock protein 90 (Hsp90) is a molecular chaperone central to client protein folding and maturation in eukaryotic cells. During its chaperone cycle, Hsp90 undergoes ATPase-coupled large-scale conformational changes between open and closed states, where the N-terminal and middle domains of the protein form a compact dimerized conformation. However, the molecular principles of the switching motion between the open and closed states remain poorly understood. Here we show by integrating atomistic and coarse-grained molecular simulations with small-angle X-ray scattering experiments and NMR spectroscopy data that Hsp90 exhibits rich conformational dynamics modulated by the charged linker, which connects the N-terminal with the middle domain of the protein. We show that the dissociation of these domains is crucial for the conformational flexibility of the open state, with the separation distance controlled by a ß-sheet motif next to the linker region. Taken together, our results suggest that the conformational ensemble of Hsp90 comprises highly extended states, which could be functionally crucial for client processing.
Asunto(s)
Proteínas HSP90 de Choque Térmico , Chaperonas Moleculares , Proteínas HSP90 de Choque Térmico/metabolismo , Modelos Moleculares , Chaperonas Moleculares/metabolismo , Simulación de Dinámica Molecular , Conformación Proteica , Pliegue de ProteínaRESUMEN
Post-translational modification of proteins with ubiquitin and ubiquitin-like proteins (Ubls) is central to the regulation of eukaryotic cellular processes. Our ability to study the effects of ubiquitylation, however, is limited by the difficulty to prepare homogenously modified proteins in vitro and by the impossibility to selectively trigger specific ubiquitylation events in living cells. Here we combine genetic-code expansion, bioorthogonal Staudinger reduction and sortase-mediated transpeptidation to develop a general tool to ubiquitylate proteins in an inducible fashion. The generated ubiquitin conjugates display a native isopeptide bond and bear two point mutations in the ubiquitin C terminus that confer resistance toward deubiquitinases. Nevertheless, physiological integrity of sortase-generated diubiquitins in decoding cellular functions via recognition by ubiquitin-binding domains is retained. Our approach allows the site-specific attachment of Ubls to nonrefoldable, multidomain proteins and enables inducible and ubiquitin-ligase-independent ubiquitylation of proteins in mammalian cells, providing a powerful tool to dissect the biological functions of ubiquitylation with temporal control.
Asunto(s)
Ingeniería de Proteínas/métodos , Ubiquitinación/genética , Ubiquitinación/fisiología , Código Genético , Unión Proteica , Procesamiento Proteico-Postraduccional/genética , Proteínas , Especificidad por Sustrato/genética , Sumoilación/genética , Ubiquitina , UbiquitinasRESUMEN
In biology, self-assembly of proteins and energy-consuming reaction cycles are intricately coupled. For example, tubulin is activated and deactivated for assembly by a guanosine triphosphate (GTP)-driven reaction cycle, and the emerging microtubules catalyze this reaction cycle by changing the microenvironment of the activated tubulin. Recently, synthetic analogs of chemically fueled assemblies have emerged, but examples in which assembly and reaction cycles are reciprocally coupled remain rare. In this work, we report a peptide that can be activated and deactivated for self-assembly. The emerging assemblies change the microenvironment of their building blocks, which consequently accelerate the rates of building block deactivation and reactivation. We quantitatively understand the mechanisms at play, and we are thus able to tune the catalysis by molecular design of the peptide precursor.
RESUMEN
Designed peptides derived from the islet amyloid polypeptide (IAPP) cross-amyloid interaction surface with Aß (termed interaction surface mimics or ISMs) have been shown to be highly potent inhibitors of Aß amyloid self-assembly. However, the molecular mechanism of their function is not well understood. Using solution-state and solid-state NMR spectroscopy in combination with ensemble-averaged dynamics simulations and other biophysical methods including TEM, fluorescence spectroscopy and microscopy, and DLS, we characterize ISM structural preferences and interactions. We find that the ISM peptide R3-GI is highly dynamic, can adopt a ß-like structure, and oligomerizes into colloid-like assemblies in a process that is reminiscent of liquid-liquid phase separation (LLPS). Our results suggest that such assemblies yield multivalent surfaces for interactions with Aß40. Sequestration of substrates into these colloid-like structures provides a mechanistic basis for ISM function and the design of novel potent anti-amyloid molecules.
Asunto(s)
Péptidos beta-Amiloides/antagonistas & inhibidores , Polipéptido Amiloide de los Islotes Pancreáticos/química , Fragmentos de Péptidos/antagonistas & inhibidores , Péptidos/química , Secuencia de Aminoácidos , Péptidos beta-Amiloides/metabolismo , Microscopía Fluorescente , Simulación de Dinámica Molecular , Resonancia Magnética Nuclear Biomolecular , Fragmentos de Péptidos/metabolismo , Péptidos/metabolismo , Especificidad por SustratoRESUMEN
Complex I functions as a redox-driven proton pump in aerobic respiratory chains. By reducing quinone (Q), complex I employs the free energy released in the process to thermodynamically drive proton pumping across its membrane domain. The initial Q reduction step plays a central role in activating the proton pumping machinery. In order to probe the energetics, dynamics, and molecular mechanism for the proton-coupled electron transfer process linked to the Q reduction, we employ here multiscale quantum and classical molecular simulations. We identify that both ubiquinone (UQ) and menaquinone (MQ) can form stacking and hydrogen-bonded interactions with the conserved Q-binding-site residue His-38 and that conformational changes between these binding modes modulate the Q redox potentials and the rate of electron transfer (eT) from the terminal N2 iron-sulfur center. We further observe that, while the transient formation of semiquinone is not proton-coupled, the second eT process couples to a semiconcerted proton uptake from conserved tyrosine (Tyr-87) and histidine (His-38) residues within the active site. Our calculations indicate that both UQ and MQ have low redox potentials around -260 and -230 mV, respectively, in the Q-binding site, respectively, suggesting that release of the Q toward the membrane is coupled to an energy transduction step that could thermodynamically drive proton pumping in complex I.
Asunto(s)
Complejo I de Transporte de Electrón/metabolismo , Electrones , Protones , Quinonas/metabolismo , Transporte de Electrón , Enlace de Hidrógeno , Modelos Moleculares , Oxidación-Reducción , Ubiquinona/metabolismo , Vitamina K 2/metabolismoRESUMEN
Metadynamic metainference has been recently introduced as a theoretical framework to determine structural ensembles by combining and weighting their noise multiple sources of experimental data with molecular mechanics force fields and metadynamics simulations. Here we build upon these initial developments to further extend and streamline the computational approach. We also show that metadynamic metainference can actually determine a structural ensemble for a disordered peptide that is essentially independent from the employed force field. We further show that it is possible to use a very computationally efficient implicit solvent force field in the place of very expensive state-of-the-art explicit solvent ones without a significant loss in accuracy.
Asunto(s)
Teorema de Bayes , Modelos Químicos , Péptidos/química , Secuencia de Aminoácidos , Simulación de Dinámica Molecular , Resonancia Magnética Nuclear Biomolecular , Oligopéptidos/química , Conformación ProteicaRESUMEN
Bacterial microcompartments (BMCs) are self-assembling, selectively permeable protein shells that encapsulate enzymes to enhance catalytic efficiency of segments of metabolic pathways through means of confinement. The modular nature of BMC shells' structure and assembly enables programming of shell permeability and underscores their promise in biotechnology engineering efforts for applications in industry, medicine, and clean energy. Realizing this potential requires methods for encapsulation of abiotic molecules, which have been developed here for the first time. We report in vitro cargo loading of BMC shells with ruthenium photosensitizers (RuPS) by two approachesâone involving site-specific covalent labeling and the other driven by diffusion, requiring no specific interactions between cargo molecules and shell proteins. The highly stable shells retain encapsulated cargo over 1 week without egress and preserve RuPS photophysical activity. This study is an important foundation for further work that will converge biological BMC architecture with synthetic chemistry to facilitate biohybrid photocatalysis.
Asunto(s)
Fármacos Fotosensibilizantes , Fármacos Fotosensibilizantes/química , Fármacos Fotosensibilizantes/metabolismo , Rutenio/químicaRESUMEN
Recent breakthroughs in neural network-based structure prediction methods, such as AlphaFold2 and RoseTTAFold, have dramatically improved the quality of computational protein structure prediction. These models also provide statistical confidence scores that can estimate uncertainties in the predicted structures, but it remains unclear to what extent these scores are related to the intrinsic conformational dynamics of proteins. Here, we compare AlphaFold2 prediction scores with explicit large-scale molecular dynamics simulations of 28 one- and two-domain proteins with varying degrees of flexibility. We demonstrate a strong correlation between the statistical prediction scores and the explicit motion derived from extensive atomistic molecular dynamics simulations and further derive an elastic network model based on the statistical scores of AlphFold2 (AF-ENM), which we benchmark in combination with coarse-grained molecular dynamics simulations. We show that our AF-ENM method reproduces the global protein dynamics with improved accuracy, providing a powerful way to derive effective molecular dynamics using neural network-based structure prediction models.
Asunto(s)
Simulación de Dinámica Molecular , Proteínas , Proteínas/química , Redes Neurales de la Computación , Conformación Molecular , Movimiento (Física) , Conformación ProteicaRESUMEN
The inherent flexibility of intrinsically disordered proteins (IDPs) makes it difficult to interpret experimental data using structural models. On the other hand, molecular dynamics simulations of IDPs often suffer from force-field inaccuracies, and long simulation times or enhanced sampling methods are needed to obtain converged ensembles. Here, we apply metainference and Bayesian/Maximum Entropy reweighting approaches to integrate prior knowledge of the system with experimental data, while also dealing with various sources of errors and the inherent conformational heterogeneity of IDPs. We have measured new SAXS data on the protein α-synuclein, and integrate this with simulations performed using different force fields. We find that if the force field gives rise to ensembles that are much more compact than what is implied by the SAXS data it is difficult to recover a reasonable ensemble. On the other hand, we show that when the simulated ensemble is reasonable, we can obtain an ensemble that is consistent with the SAXS data, but also with NMR diffusion and paramagnetic relaxation enhancement data.
RESUMEN
Small-angle X-ray scattering (SAXS) experiments provide low-resolution but valuable information about the dynamics of biomolecular systems, which could be ideally integrated into molecular dynamics (MD) simulations to accurately determine conformational ensembles of flexible proteins. The applicability of this strategy is hampered by the high computational cost required to calculate scattering intensities from three-dimensional structures. We previously presented a hybrid resolution method that makes atomistic SAXS-restrained MD simulation feasible by adopting a coarse-grained approach to efficiently back-calculate scattering intensities; here, we extend this technique, applying it in the framework of metainference with the aim to investigate the dynamical behavior of flexible biomolecules. The efficacy of the method is assessed on the K63-diubiquitin, showing that the inclusion of SAXS restraints is effective in generating a reliable conformational ensemble, improving the agreement with independent experimental data.
Asunto(s)
Simulación de Dinámica Molecular , Dispersión del Ángulo Pequeño , Ubiquitinas/química , Difracción de Rayos X , Conformación ProteicaRESUMEN
Polyubiquitin chains are flexible multidomain proteins, whose conformational dynamics enable them to regulate multiple biological pathways. Their dynamic is determined by the linkage between ubiquitins and by the number of ubiquitin units. Characterizing polyubiquitin behavior as a function of their length is hampered because of increasing system size and conformational variability. Here, we introduce a new approach to efficiently integrating small-angle x-ray scattering with simulations allowing us to accurately characterize the dynamics of linear di-, tri-, and tetraubiquitin in the free state as well as of diubiquitin in complex with NEMO, a central regulator in the NF-κB pathway. Our results show that the behavior of the diubiquitin subunits is independent of the presence of additional ubiquitin modules and that the dynamics of polyubiquitins with different lengths follow a simple model. Together with experimental data from multiple biophysical techniques, we then rationalize the 2:1 NEMO:polyubiquitin binding.
RESUMEN
Respiratory complex I (NADH:ubiquinone oxidoreductase) captures the free energy from oxidising NADH and reducing ubiquinone to drive protons across the mitochondrial inner membrane and power oxidative phosphorylation. Recent cryo-EM analyses have produced near-complete models of the mammalian complex, but leave the molecular principles of its long-range energy coupling mechanism open to debate. Here, we describe the 3.0-Å resolution cryo-EM structure of complex I from mouse heart mitochondria with a substrate-like inhibitor, piericidin A, bound in the ubiquinone-binding active site. We combine our structural analyses with both functional and computational studies to demonstrate competitive inhibitor binding poses and provide evidence that two inhibitor molecules bind end-to-end in the long substrate binding channel. Our findings reveal information about the mechanisms of inhibition and substrate reduction that are central for understanding the principles of energy transduction in mammalian complex I.
Asunto(s)
Complejo I de Transporte de Electrón/química , Complejo I de Transporte de Electrón/metabolismo , Inhibidores Enzimáticos/metabolismo , Mamíferos/metabolismo , Animales , Sitios de Unión , Microscopía por Crioelectrón , Complejo I de Transporte de Electrón/antagonistas & inhibidores , Complejo I de Transporte de Electrón/genética , Inhibidores Enzimáticos/química , Femenino , Mamíferos/genética , Ratones , Ratones Endogámicos C57BL , Mitocondrias Cardíacas/genética , Mitocondrias Cardíacas/metabolismo , Simulación de Dinámica Molecular , Fosforilación Oxidativa , Piridinas/química , Piridinas/metabolismoRESUMEN
Cardiolipin modulates the activity of membrane-bound respiratory enzymes that catalyze biological energy transduction. The respiratory complex I functions as the primary redox-driven proton pump in mitochondrial and bacterial respiratory chains, and its activity is strongly enhanced by cardiolipin. However, despite recent advances in the structural biology of complex I, cardiolipin-specific interaction mechanisms currently remain unknown. On the basis of millisecond molecular simulations, we suggest that cardiolipin binds to proton-pumping subunits of complex I and induces global conformational changes that modulate the accessibility of the quinone substrate to the enzyme. Our findings provide key information on the coupling between complex I dynamics and activity and suggest how biological membranes modulate the structure and activity of proteins.
Asunto(s)
Cardiolipinas/química , Complejo I de Transporte de Electrón/química , Conformación Proteica , Subunidades de Proteína/química , Sitios de Unión , Membrana Celular/química , Cristalografía por Rayos X , Complejo I de Transporte de Electrón/genética , Metabolismo Energético/genética , Membrana Dobles de Lípidos/química , Mitocondrias/química , Mitocondrias/metabolismo , Simulación de Dinámica Molecular , Oxidación-Reducción , Unión Proteica , Bombas de Protones/química , Relación Estructura-Actividad , Thermus thermophilus/químicaRESUMEN
Post-transcriptional mechanisms play a predominant role in the control of microRNA (miRNA) production. Recognition of the terminal loop of precursor miRNAs by RNA-binding proteins (RBPs) influences their processing; however, the mechanistic basis for how levels of individual or subsets of miRNAs are regulated is mostly unexplored. We previously showed that hnRNP A1, an RBP implicated in many aspects of RNA processing, acts as an auxiliary factor that promotes the Microprocessor-mediated processing of pri-mir-18a. Here, by using an integrative structural biology approach, we show that hnRNP A1 forms a 1:1 complex with pri-mir-18a where both RNA recognition motifs (RRMs) bind to cognate RNA sequence motifs in the terminal loop of pri-mir-18a. Terminal loop binding induces an allosteric destabilization of base-pairing in the pri-mir-18a stem that promotes its downstream processing. Our results highlight terminal loop RNA recognition by RBPs as a potential general principle of miRNA biogenesis and regulation.
Asunto(s)
Ribonucleoproteína Nuclear Heterogénea A1/química , Ribonucleoproteína Nuclear Heterogénea A1/metabolismo , MicroARNs/química , MicroARNs/metabolismo , Secuencia de Bases , Sitios de Unión , Fenómenos Biofísicos , Cristalografía por Rayos X , Células HeLa , Ribonucleoproteína Nuclear Heterogénea A1/genética , Humanos , MicroARNs/genética , Modelos Moleculares , Simulación de Dinámica Molecular , Resonancia Magnética Nuclear Biomolecular , Conformación de Ácido Nucleico , Unión Proteica , Dominios Proteicos , Procesamiento Postranscripcional del ARN , Estabilidad del ARNRESUMEN
Membrane-assisted amyloid formation is implicated in human diseases, and many of the aggregating species accelerate amyloid formation and induce cell death. While structures of membrane-associated intermediates would provide tremendous insights into the pathology and aid in the design of compounds to potentially treat the diseases, it has not been feasible to overcome the challenges posed by the cell membrane. Here, we use NMR experimental constraints to solve the structure of a type-2 diabetes related human islet amyloid polypeptide intermediate stabilized in nanodiscs. ROSETTA and MD simulations resulted in a unique ß-strand structure distinct from the conventional amyloid ß-hairpin and revealed that the nucleating NFGAIL region remains flexible and accessible within this isolated intermediate, suggesting a mechanism by which membrane-associated aggregation may be propagated. The ability of nanodiscs to trap amyloid intermediates as demonstrated could become one of the most powerful approaches to dissect the complicated misfolding pathways of protein aggregation.