Mechanism of eIF6 release from the nascent 60S ribosomal subunit

Abstract

SBDS protein (deficient in the inherited leukemia-predisposition disorder Shwachman-Diamond syndrome) and the GTPase EFL1 (an EF-G homolog) activate nascent 60S ribosomal subunits for translation by catalyzing eviction of the antiassociation factor eIF6 from nascent 60S ribosomal subunits. However, the mechanism is completely unknown. Here, we present cryo-EM structures of human SBDS and SBDS-EFL1 bound to Dictyostelium discoideum 60S ribosomal subunits with and without endogenous eIF6. SBDS assesses the integrity of the peptidyl (P) site, bridging uL16 (mutated in T-cell acute lymphoblastic leukemia) with uL11 at the P-stalk base and the sarcin-ricin loop. Upon EFL1 binding, SBDS is repositioned around helix 69, thus facilitating a conformational switch in EFL1 that displaces eIF6 by competing for an overlapping binding site on the 60S ribosomal subunit. Our data reveal the conserved mechanism of eIF6 release, which is corrupted in both inherited and sporadic leukemias.

Figure 1

SBDS shields the active sites of the 60S subunit. (a,b) Crown view (a) and transverse section (b) of the cryo-EM map of the 60S–eIF6–SBDS complex, filtered to 4 Å. The 60S ribosomal subunit is shown in cyan, eIF6 in yellow and SBDS in magenta. CP, central protuberance; SB, stalk base; PTC, peptidyl transferase center; N, N terminus. (c–e) Atomic models of the interface between the 60S ribosomal subunit and eIF6 (c), SBDS domain I (d) and SBDS domain III (e). 26S rRNA is shown in blue, ribosomal proteins in beige, eIF6 in yellow and SBDS in magenta. Residues R98 and M123 (Q123 in humans) of human uL16 that are mutated in T-ALL, are indicated. SRL, sarcin-ricin loop.

Figure 2

EFL1 and eIF6 compete for an overlapping binding site. (a,b) Crown views of the cryo-EM maps of the 60S–eIF6–SBDS–EFL1 (a) and 60S–SBDS–EFL1 (b) complexes, filtered to 6 Å. EFL1 is in dark blue. CP, central protuberance; SB, stalk base. (c) Superposition of the cryo-EM densities, filtered to 6 Å (top) or atomic models (bottom) of EFL1 in the presence (gray) or absence (dark blue) of eIF6. (d) The volume previously occupied by eIF6, highlighted in yellow mesh in the 60S–SBDS–EFL1 cryo-EM map. (e,f) Atomic models of the 60S–SBDS–EFL1 complex with (e) or without (f) eIF6. 26S rRNA is in blue, ribosomal proteins in beige, SBDS in magenta, EFL1 domain I in orange, EFL1 domains II–V in dark blue and eIF6 in yellow.

Figure 3

Rotational displacement of SBDS upon EFL1 binding. (a–c) Top views of 60S–eIF6–SBDS (a), 60S–eIF6–SBDS–EFL1 (b) and 60S–SBDS–EFL1 (c) complexes. SBDS is shown in magenta, the 60S subunit in cyan, eIF6 in yellow and EFL1 in dark blue. For clarity, 60S, eIF6 and EFL1 densities are shown in transparency. The uL1 protein stalk (uL1), central protuberance (CP) and P-stalk base (SB) are indicated. (d) Superposition of the SBDS structures from the 60S–eIF6–SBDS (purple), 60S–eIF6-SBDS–EFL1 (red) and 60S–SBDS–EFL1 (black) complexes. SBDS helix α5 is indicated.

Figure 4

Disease-related SBDS variants disrupt critical interactions with the 60S rRNA. (a) Complementation of sdo1Δ cells by disease-related SDO1 variant alleles. Ten-fold serial dilutions (from left to right) of the indicated strains are shown. 5-FOA, 5-fluoroorotic acid. (b) Impaired 60S-subunit binding of disease-related Sdo1 variants in vivo. FLAG-tagged Sdo1 was visualized in the supernatant (S) and pellet (P), and uL16 was visualized in the pellet across the indicated range of NaCl concentrations by immunoblotting. (c–e) Mapping disease-related SBDS residues in the 60S–eIF6–SBDS–EFL1 atomic model including K67 (c), K151 and R218 (d) and Q94-V95 and D97-K98 (e).

Figure 5

Mechanism of eIF6 release by SBDS and EFL1. (a) SBDS (closed state) is recruited to a late cytoplasmic eIF6-loaded pre-60S subunit after P-stalk base assembly and uL16 recruitment. (b) EFL1–GTP (or EF-2–GTP in archaea) binds directly to SBDS and eIF6 in the GTPase center, thus promoting rotational displacement (180°) of SBDS domain III away from the P-stalk base toward helix 69 (open state), which is stabilized by SBDS residues K151 and R218. (c) GTP-bound EFL1 in the accommodated state competes with eIF6 for an overlapping binding site on the 60S subunit, thus promoting eIF6 displacement. (d) Interaction of EFL1–GTP with the SRL promotes GTP hydrolysis, thus triggering a conformational switch in EFL1 that promotes a low-affinity ribosome binding state. SBDS and EFL1–GDP dissociate from the 60S subunit. (e–h) Eukaryotic ribosome maturation is structurally reminiscent of prokaryotic ribosome recycling. Atomic models of human SBDS (left) from the 60S–eIF6–SBDS complex, ribosome-recycling factor (RRF) from Thermus thermophilus (right) (PDB 3J0D) (e) and density maps of SBDS (left) and RRF (right) bound to the large ribosomal subunit in the absence (f) or presence (g,h) of EFL1 (left) or EF-G (right). The 60S subunit (60S, left; 50S, right) is in cyan, SBDS (domains I–III) and RRF (I and II) in purple, EFL1 (I–V) and EF-G (I–V) in dark blue, eIF6 in yellow. uL1, uL1 protein stalk; CP, central protuberance; SB, P-stalk base; bL12, bL12 protein stalk. Density maps for the 50S–RRF and 50S–RRF–EFG complexes were generated from PDB 3J0D and PDB 2RDO with IMAGIC-V.