The essential ATP-binding cassette protein ABCE1 splits 80S ribosomes into 60S and 40S subunits after canonical termination or quality-control-based mRNA surveillance processes. However, the underlying splitting mechanism remains enigmatic. Here, we present a cryo-EM structure of the yeast 40S–ABCE1 post-splitting complex at 3.9-Å resolution. Compared to the pre-splitting state, we observe repositioning of ABCE1's iron-sulfur cluster domain, which rotates 150° into a binding pocket on the 40S subunit. This repositioning explains a newly observed anti-association activity of ABCE1. Notably, the movement implies a collision with A-site factors, thus explaining the splitting mechanism. Disruption of key interactions in the post-splitting complex impairs cellular homeostasis. Additionally, the structure of a native post-splitting complex reveals ABCE1 to be part of the 43S initiation complex, suggesting a coordination of termination, recycling, and initiation.
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The authors thank K. Kiosze-Becker, E. Nürenberg-Goloub, B. Hetzert, C. Le Gal and C. Thomas for helpful suggestions on the manuscript and C. Ungewickell, S. Lange and S. Lamberth for technical assistance. This work was supported by the German Research Council (grants SFB 902 to R.T., SFB 646 to R.B. and T.B., FOR 1805 to R.B., GRK 1721 to R.B.). R.B. acknowledges support by the Center for Integrated Protein Science Munich (CiPS-M) and the European Research Council (Advanced Grants CRYOTRANSLATION). The Cluster of Excellence–Macromolecular Complexes (EXC 115 to R.T.) supported the work. M.G. and C.S. were supported by Boehringer Ingelheim Fonds PhD fellowships. We thank the Leibniz-Rechenzentrum Munich (LRZ) for providing computational services and support.
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In the free state, ABCE1 is in the open state with two ADPs occluded. After binding to 80S pre-termination complexes (with eRF1 or Dom34 or Pelota), ABCE1 changes its conformation to an intermediate, semi-closed (pre-splitting) state. Here the nucleotide occupation is less clear. High and medium resolved cryo-EM structures showed density for a bound nucleotide in NBS I, whereas NBS II was rather empty. Occlusion of both NBSs with ATP leads to closure of the NBDs which is required for ribosome splitting. This closure is accompanied by rearrangement of the FeS cluster domain. In the post-splitting state, ABCE1 can be trapped on the 40S subunit in a closed conformation occluding two ATP (here AMP-PNP). The FeS cluster domain rotates into a binding cleft formed by h5, h44 and uS12. This conformation is mainly stabilized by interactions of Pro30 to uS12 and Arg7 to h5. Moreover, the cantilever helix unwinds and Tyr301 contacts the backbone of Asn78. The HLH motif interacts with the junction of h8 and h14 mainly via Ser150. Additional conformational changes occur in the C-terminal helix of eS24, which contacts NBD1 (Gln262) in post-splitting state. Only minor conformational changes occur for the hinge 2 motif, which contacts the h5 and h15 junction via Arg573 and Ser588. A zoom into NBS I shows how the nucleotide is occluded by ABC motifs. Open (homology model based on PDB: 3BK7), semi-closed (PDB: 3J16), and closed state (this study) of ABCE1 were morphed using UCSF-Chimera.
In the pre-splitting state, ABCE1 is bound to an 80S ribosome occupied with an A-site factor (here eRF1). The FeS cluster domain interacts with the C-terminal domain of eRF1 (or Pelota). In phase 1, ATP-binding leads to closing of the NBDs and rearrangement of the FeS cluster domain in direction of the bound A-site factor. It acts as a wedge, which leads to destabilization of the 80S ribosome. In phase 2, after dissociation of the 60S, the FeS cluster domain locks into its final position and prevents formation of an intersubunit bridge B5 involving uL14 (green), thus acting as an anti-association factor. Isolated densities of the 60S subunit (dark grey), eRF1 (blue), pre-splitting state of ABCE1 (yellow) as well as respective models were taken from EMD-2598 (PDB: 4CRM).
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