ABCE1 Controls Ribosome Recycling by an Asymmetric Dynamic Conformational Equilibrium

The twin-ATPase ABCE1 has a vital function in mRNA translation by recycling terminated or stalled ribosomes. As for other functionally distinct ATP-binding cassette (ABC) proteins, the mechanochemical coupling of ATP hydrolysis to conformational changes remains elusive. Here, we use an integrated biophysical approach allowing direct observation of conformational dynamics and ribosome association of ABCE1 at the single-molecule level. Our results from FRET experiments show that the current static two-state model of ABC proteins has to be expanded because the two ATP sites of ABCE1 are in dynamic equilibrium across three distinct conformational states: open, intermediate, and closed. The interaction of ABCE1 with ribosomes influences the conformational dynamics of both ATP sites asymmetrically and creates a complex network of conformational states. Our findings suggest a paradigm shift to redefine the understanding of the mechanochemical coupling in ABC proteins: from structure-based deterministic models to dynamic-based systems

Structure of the ribosome post-recycling complex probed by chemical cross-linking and mass spectrometry

Ribosome recycling orchestrated by the ATP binding cassette (ABC) protein ABCE1 can be considered as the final-or the first-step within the cyclic process of protein synthesis, connecting translation termination and mRNA surveillance with re-initiation. An ATP-dependent tweezer-like motion of the nucleotide-binding domains in ABCE1 transfers mechanical energy to the ribosome and tears the ribosome subunits apart. The post-recycling complex (PRC) then re-initiates mRNA translation. Here, we probed the so far unknown architecture of the 1-MDa PRC (40S/30S.ABCE1) by chemical cross-linking and mass spectrometry (XL-MS). Our study reveals ABCE1 bound to the translational factor-binding (GTPase) site with multiple cross-link contacts of the helix-loop-helix motif to the S24e ribosomal protein. Cross-linking of the FeS cluster domain to the ribosomal protein S12 substantiates an extreme lever-arm movement of the FeS cluster domain during ribosome recycling. We were thus able to reconstitute and structurally analyse a key complex in the translational cycle, resembling the link between translation initiation and ribosome recycling

The antimalarial drug primaquine targets Fe–S cluster proteins and yeast respiratory growth

Malaria is a major health burden in tropical and subtropical countries. The antimalarial drug primaquine is extremely useful for killing the transmissible gametocyte forms of Plasmodium falciparum and the hepatic quiescent forms of P. vivax. Yet its mechanism of action is still poorly understood. In this study, we used the yeast Saccharomyces cerevisiae model to help uncover the mode of action of primaquine. We found that the growth inhibitory effect of primaquine was restricted to cells that relied on respiratory function to proliferate and that deletion of SOD2 encoding the mitochondrial superoxide dismutase severely increased its effect, which can be countered by the overexpression of AIM32 and MCR1 encoding mitochondrial enzymes involved in the response to oxidative stress. This indicated that ROS produced by respiratory activity had a key role in primaquine-induced growth defect. We observed that Δsod2 cells treated with primaquine displayed a severely decreased activity of aconitase that contains a Fe–S cluster notoriously sensitive to oxidative damage. We also showed that in vitro exposure to primaquine impaired the activity of purified aconitase and accelerated the turnover of the Fe–S cluster of the essential protein Rli1. It is suggested that ROS-labile Fe–S groups are the primary targets of primaquine. Aconitase activity is known to be essential at certain life-cycle stages of the malaria parasite. Thus primaquine-induced damage of its labile Fe–S cluster – and of other ROS-sensitive enzymes – could inhibit parasite development

Cellulose to 2,5-Dimethylfuran Process

The synthesis pathway to the biofuel 2,5-dimethylfuran (DMF) from cellulose involves acidcatalyzed, mechanocatalytic depolymerization of cellulose to glucose, Lewis acid catalyzed isomerization of glucose to fructose, Brønsted acid-catalyzed dehydration of fructose to 5-hydroxymethylfurfural (HMF), and finally the hydrodeoxygenation of HMF over a noble metal alloy catalyst deposited on carbonaceous support. A one-pot reaction system to produce HMF directly from glucose avoiding the isolation of fructose is developed using solely Sn-incorporated zeolite of the beta structure as catalyst under an elevated temperature of 180°C in a mixture of water and ethanol in the ratio of 80 to 20. The best-obtained yield of HMF is 36 %. In the second part of the study, purified HMF is used to produce DMF over a PtCo alloy catalyst supported on graphitic carbon. A major aim is to avoid separation and purification by choosing a solvent, which might be used as a fuel blended with DMF. Among a variety of solvents studied, commercial gasoline is found to be well suited for this reaction. The reaction in gasoline is conducted in a sequential manner up to three times with an optimized initial loading of 10 wt% HMF per step, resulting in a concentration increase of up to 7 wt% DMF for each conversion step, by which a concentration range between 7 and 20 wt% DMF in the final blend is covered. Best results are obtained with gasoline:ethanol mixtures in the ratio 9:1, commonly known as E10, as ethanol is found to act as a solvent mediator for the dissolution of HMF in the reaction system. The fuel properties of the resulting fuel blend are accessed by measuring the derived cetane number (DCN) and the distillation profile and found to be comparable with reference blends. This makes final purification obsolete and leads to a biofuel blend potentially usable in current internal combustion engines

Molecular mechanism of the ribosome recycling factor ABCE1

Protein biosynthesis is a conserved process, essential for life. Proteins are assembled from single amino acids according to their genetic blueprint in the form of a messenger ribonucleic acid (mRNA). Peptide bond formation is catalyzed by ancient ribonucleic acid (RNA) residues within the supramolecular ribosomal complex, which is organized in two dynamic subunits (Ramakrishnan, 2014). Each subunit comprises large ribosomal RNA (rRNA) molecules and several dozens of peripheral proteins. mRNA translation has been divided into three phases, namely translation initiation, elongation and termination in biochemistry textbooks. During initiation, the ribosomal subunits assemble into a functional ribosome on an activated mRNA and acquire the first transfer RNA (tRNA), an adapter between the start codon on the mRNA and the N-terminal methionine of the protein (Hinnebusch and Lorsch, 2012). During elongation, the ribosome translocates along the mRNA exposing one codon after the other, and amino acids are delivered to the ribosome by the respective tRNAs, and attached to the nascent polypeptide chain. During termination, the polypeptide is released and the ribosome remains loaded with mRNA and tRNA at the end of the open reading frame for the translated gene (Hellen, 2018). Bacterial ribosomes are subsequently recycled by a specific ribosome recycling factor and the small ribosomal subunit is simultaneously consigned to initiation factors for a next round of translation – rendering bacterial translation as a cyclic process with an additional ribosome recycling phase. However, the process of ribosome recycling remained enigmatic in Eukarya and Archaea until the simultaneous discovery of the twin-ATPase ABCE1 as the major ribosome recycling factor. Strikingly, ABCE1 has initially been shown to participate in translation initiation (Nürenberg and Tampé, 2013). Thus, closing the translation cycle by revealing the detailed molecular mechanism of ABCE1 and its role for translation initiation are the two goals of this research. Beyond the plenitude of well-studied translational GTPases, ABCE1 is the only essential factor energized by ATP, delivering the energy for ribosome splitting via two nucleotide-binding sites. Here, I define how allosterically coupled ATP binding and hydrolysis events in ABCE1 empower ribosome recycling. ATP occlusion in the low-turnover control site II promotes formation of the pre-splitting complex and facilitates ATP engagement in the high-turnover site I, which in turn drives the structural re- organization required for ribosome splitting. ATP hydrolysis and ensuing release of ABCE1 from the small subunit terminate the post-splitting complex. Thus, ABCE1 runs through an allosterically coupled cycle of closure and opening at both sites consistent with a processive clamp model. This study delineates the inner mechanics of ABCE1 and reveals why various ABCE1 mutants lead to defects in cell homeostasis, growth, and differentiation (Nürenberg-Goloub et al., 2018). Additionally, a high-resolution cryo-electron microscopy (EM) structure of the archaeal post-splitting complex was obtained, revealing a central macromolecular assembly at the crossover of ribosome recycling and translation initiation. Conserved interactions between ABCE1 and the small ribosomal subunit resemble the eukaryotic complex (Heuer et al., 2017). The conformational state of ABCE1 at the post-splitting complex confirms the molecular mechanism of ribosome recycling uncovered in this study. Moving further along the reaction coordinate of cellular translation, I reconstitute the complete archaeal translation initiation pathway and show that essential archaeal initiation factors are recruited to the post-splitting complex by biochemical methods and cryo-EM structures at intermediate resolution. Thus, the archaeal translation cycle is closed, following its bacterial model and paving the way for a deeper understanding of protein biosynthesis

Cellulose to 2,5-dimethylfuran process

Bei der Herstellung von 5-Hydroxymethylfurfural (HMF) aus Glukose wird unter Verwendung von Sn-BEA-Katalysator eine HMF-Ausbeute von 36 % erreicht. Im zweiten Teil der Studie wird die Hydrodeoxygenierung von HMF zu 2,5-Dimethylfuran (DMF) über einen PtCo-Katalysator unter Wasserstoffdruck durchgeführt. Es werden Lösungsmittel verwendet, welche als Vergasertreibstoffe in Frage kommen, um eine Aufreinigung von DMF zu vermeiden. Eine Mischung aus Benzin und Ethanol ist für diese Reaktion gut geeignet. Die Reaktion im Benzin wird sequentiell bis zu dreimal mit einer Anfangsbeladung von 10 Gew.-% HMF pro Schritt durchgeführt, wodurch die Mischung mit bis zu 20 Gew.-% DMF angereichert wird. Die Klopfeigenschaften und das Destillationsprofil des resultierenden Kraftstoffgemischs werden ermittelt und sind mit Referenzmischungen vergleichbar. Dies macht die Endreinigung überflüssig und führt zu einer Biokraftstoffmischung, die potenziell in aktuellen Verbrennungsmotoren einsetzbar ist