4 research outputs found
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
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
The motor protein ABCE1 drives the essential process of ribosome recycling
Die Proteinbiosynthese (Translation) ist ein fĂŒr alle Lebewesen zentraler Prozess, bei dem in mehreren Schritten die genetische Information der DNA in die AminosĂ€uresequenz eines Proteins ĂŒbersetzt wird. Die Translation setzt sich aus vier Phasen zusammen, der Initiation, der Elongation, der Termination und dem RibosomâRecycling. Dem Recycling der Ribosomen kommt dabei eine entscheidende SchlĂŒsselfunktion zu, da es die Termination mit einer neu eingeleiteten Initiation verbindet und somit den Prozess der Proteinbiosynthese zu einem sich wiederholenden Zyklus macht. Der Prozess des Recyclings wird hierbei hauptsĂ€chlich von dem ATPâbindenden Protein ABCE1 umgesetzt, welches das Ribosom (80S/70S) in seine groĂe (60S/50S) und kleine (40S/30S) Untereinheit spaltet. RibosomâRecycling findet dabei nicht nur innerhalb eines korrekt ablaufenden Zyklus der Proteinbiosynthese statt, sondern auch an leeren Ribosomen sowie wĂ€hrend der RibosomenâBiogenese und sogenannter mRNA surveillanceâVorgĂ€nge, wenn die Translationsmaschinerie z.B. auf Grund von sekundĂ€ren Strukturen der mRNA unwiderruflich gestoppt wird...Protein synthesis is a central process within every living cell, where information embodied in the nucleotide sequence of the mRNA is translated into the primary sequence of proteins. The translation procedure comprises four steps: initiation, elongation, termination, and recycling. Ribosome recycling orchestrated by the ATPâbinding cassette (ABC) protein ABCE1, renders mRNA translation into a cyclic process, connecting termination with re initiation. In Archaea and Eukarya, the ABC protein ABCE1 catalyzes ribosome recycling by splitting the ribosome (80S/70S) into the small 40S/30S and large 60S/50S subunits, providing them for the next translation round.
The ABCâtype ATPase one of the most conserved proteins, present in all Archaea and Eukarya, but not in Bacteria, is essential for life in all organisms examined so far. ABCE1 was initially identified as RNase L inhibitor (Rli1), involved in the antiviral RNA immunity, and as host protein 68 (HP68) playing a role in HIV capsid assembly. However, the strong sequence conservation of ABCE1 points towards a more fundamental function within cell homeostasis, which was found by its involvement in various translation processes. ABCE1 turned out to be the major ribosome recycling factor indispensable for life in Eukarya and Archaea, being involved in canonical translation, mRNA surveillance, ribosome biogenesis, and translation initiation.
Recent functional and structural data provided first insights into the mechanism of ABCE1 in ribosome recycling. The nucleotideâbinding domains (NBDs) sandwich two ATP molecules in the NBD1âNBD2 interface causing an NBD engagement, which is released upon ATP hydrolysis. In case of ABCE1, this ATPâdependent tweezerâlike motion of the NBDs transfers mechanical energy to the ribosome and tears the subunits apart. The FeSâcluster domain may swing out of the NBD cleft into the interâsubunit space of the ribosome, which drives the subunits apart either directly or via the bound a/eRF1. Hence, the subunits are released and the postâsplitting complex (PSC, 40S/30SâABCE1âATP) is available for reâinitiation events, presumably occurring via the known interactions of ABCE1with initiation factors.
One of the most crucial aspects of this model is the nucleotideâdependent conformational switch of ABCE1, which drives ribosomal subunit splitting. However, the conformational states, which ABCE1 undergoes during ribosome recycling, including their mechanistic importance for its diverse functions, remain unknown. Further, the exact role and movement of the essential FeScluster domain during ribosome recycling are not yet understood. Additional, it remains elusive where ABCE1 is bound in the postâsplitting complex and how the splitting mechanism is regulated concerning the asymmetric NBDs and the coupling of nucleotide binding with NBD closing and ATP hydrolysis.
Thus, in order to monitor the conformational dynamics of the ribosome recycling factor ABCE1 two complementing methods in structural biology, namely singleâmolecule based Förster resonance energy transfer (smFRET) and pulsed electronâelectron double resonance (PELDOR) spectroscopy were applied.
Singleâmolecule FRET as an integrated biophysical approach based on Förster resonance energy transfer and singleâmolecule detection was used to understand the fundamental molecular principles of ABCE1. Contrary to the anticipated twoâstate model of ABC proteins, it was shown in this thesis that both nucleotideâbinding sites of ABCE1 are always in a dynamic equilibrium between conformational states with distinct properties: open, intermediate, and closed. The equilibrium in the two nucleotideâbinding sites is distinctly affected when ABCE1 interacts with ribosomal subunits and nucleotides. While ABCE1 can adopt all three conformational states in its free or 30S bound situation, the closed state has the highest affinity for 30S subunit. Further, dissociation of ABCE1 from the small ribosomal subunit, a step that completes the recycling process, is followed by the opening of the NBSs. Hence, the current findings have important implications not only for ribosome recycling but represent a new paradigm for the molecular mechanisms of twinâATPases.
The complementing PELDOR measurements provide the advantage of high distance precision and reliability studying macromolecular complexes. Distance distributions of a number of ABCE1 variants even bound to the 1âMDa postâsplitting complex (30SâABCE1âAMPâPNP), composed of the 16S rRNA, 28 ribosomal proteins, and ABCE1, was analyzed. Thus, the available crystal structures of ABCE1 in the open state were validated, since all distances of ABCE1 measured in this study perfectly correspond to this crystallized state. Unfortunately, ABCE1 could not be trapped in the closed state under the experimental conditions applied, although plenty different approaches to stabilize this state were performed.
In the second part of this study the architecture yet unknown of the 1âMDa post splitting complex (40S/30SâABCE1âATP), concerning especially the ABCE1 binding site and its interactions with translational proteins, was probed by a method, which combines chemical cross linking with massâspectrometry (XLâMS). Following this approach, it was demonstrated that ABCE1 remains bound at the translational GTPaseâbinding site after ribosome splitting, contacting the S24e protein of the small subunit. The platform for the intensive contacts to the small ribosomal subunit is thereby provided by the unique helixâloopâhelix motif of ABCE1. Notably, the FeScluster domain of ABCE1 undergoes a large rotational and translational rearrangement towards the small ribosomal subunit S12 upon nucleotideâdependent closure of the NBDs. Thus, a key complex in the translational cycle, resembling the link between translation initiation and ribosome recycling processes, was reconstituted and structurally analyzed
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.status: publishe