Molecular analysis of the ribosome recycling factor ABCE1 bound to the 30S post-splitting complex.

Ribosome recycling by the twin-ATPase ABCE1 is a key regulatory process in mRNA translation and surveillance and in ribosome-associated protein quality control in Eukarya and Archaea. Here, we captured the archaeal 30S ribosome post-splitting complex at 2.8 Å resolution by cryo-electron microscopy. The structure reveals the dynamic behavior of structural motifs unique to ABCE1, which ultimately leads to ribosome splitting. More specifically, we provide molecular details on how conformational rearrangements of the iron-sulfur cluster domain and hinge regions of ABCE1 are linked to closure of its nucleotide-binding sites. The combination of mutational and functional analyses uncovers an intricate allosteric network between the ribosome, regulatory domains of ABCE1, and its two structurally and functionally asymmetric ATP-binding sites. Based on these data, we propose a refined model of how signals from the ribosome are integrated into the ATPase cycle of ABCE1 to orchestrate 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.

Ribosome recycling is coordinated by processive events in two asymmetric ATP sites of ABCE1.

Ribosome recycling orchestrated by ABCE1 is a fundamental process in protein translation and mRNA surveillance, connecting termination with initiation. 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 by a yet unknown mechanism. Here, we 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.