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.
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.
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.
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.
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.
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 β-strand structure distinct from the conventional amyloid β-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.
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.
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.
Synthetic lipid membranes have served as important models for cellular membranes. However, these static membranes do not recapitulate the dynamic nature of the biological membranes which are ...frequently remodeled to support cellular function. An ideal membrane model would thus also display dynamic exchange of lipids. In this work, we achieve such a system by coupling the self‐assembly of peptides into membranes with a chemical reaction cycle. The reaction cycle activates and deactivates the peptides for self‐assembly at the expense of a chemical fuel. The resulting membranes are dynamically remodeled, and, over their 40 min lifetime, they emerge, grow, and are torn apart before they eventually decay.
Keep it moving: The self‐assembly of peptides into vesicles is coupled to a dynamic chemical reaction cycle. These vesicles emerge in response to fuel, and continuously remodel and decay when all fuel is depleted.
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.
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