Mechanistic insights into the alternative ribosome recycling by HflXr

Abstract

During stress conditions such as heat shock and antibiotic exposure, ribosomes stall on messenger RNAs, leading to inhibition of protein synthesis. To remobilize ribosomes, bacteria use rescue factors such as HflXr, a homolog of the conserved housekeeping GTPase HflX that catalyzes the dissociation of translationally inactive ribosomes into individual subunits. Here we use time-resolved cryo-electron microscopy to elucidate the mechanism of ribosome recycling by Listeria monocytogenes HflXr. Within the 70S ribosome, HflXr displaces helix H69 of the 50S subunit and induces long-range movements of the platform domain of the 30S subunit, disrupting inter-subunit bridges B2b, B2c, B4, B7a and B7b. Our findings unveil a unique ribosome recycling strategy by HflXr which is distinct from that mediated by RRF and EF-G. The resemblance between HflXr and housekeeping HflX suggests that the alternative ribosome recycling mechanism reported here is universal in the prokaryotic kingdom.

Graphical Abstract

Figure 1.

Time-resolved cryo-EM of HflXr-mediated ribosome recycling. (A) Change in light scattering as a response to HflXr-mediated dissociation of 70S ribosomes (0.05 μM) at 37°C and 20°C. The scattered light intensity at 435 nm was measured in a stopped flow instrument after rapid mixing with HflXr (0.5 μM) in the presence of GDPCP (500 μM). Curves represent the average of 5–8 individual traces. (B) Relative distribution of 70S ribosomes (red) and 50S subunits (blue) over time, obtained from particle distributions in the cryo-EM datasets. (C) Cryo-EM maps of seven ribosome states captured during HflXr-mediated ribosome recycling and their assignment as pre-HflXr binding, intermediates, and post-splitting. Maps are colored to show the 50S subunit (blue), 30S subunit body domain (yellow), 30S subunit head domain (gold), HflXr (pink), HPF (purple), tRNA (green), and mRNA (cyan). (D) The NTD of HflXr (pink) bound to the cleft formed by H69 and H71 in the 50S subunit in structure II-C. In structure I-B (gray), H69 would collide with HflXr, resulting in the displacement of H69 with a concomitant movement of the platform domain of the 30S subunit away from the 50S subunit (downward red arrows).

Figure 2.

HflXr displaces H69 and h44 without disrupting bridge B2a. (A) (left) Binding of HflXr (pink) to structure II-C near bridge B2a causes displacement of H69 (white) from its position in I-B (gray), resulting in an approximately equal displacement of h44 (yellow) toward the 30S platform domain. Conserved hydrogen bond interactions in bridge B2a in the presence (center; II-C) or absence (right; I-B) of HflXr. (B) (left) Despite the presence of HPF (purple) in II-B, HflXr (pink) causes a similar movement of bridge B2a. The integrity of the bridge is maintained irrespective whether HflXr is bound (center; II-B) or not (right; I-A). (C) Binding of HflXr (pink) in II-D near bridge B2a results in approximately equal displacement of H69 (white) and h44 (yellow) from their position in II-A (gray). (D) Binding of HflXr to the 70S ribosome in structures II-B, II-C and II-D positions H69 similar to that in the rotated state II-A without HflXr.

Figure 3.

HflXr induces conformational changes in the platform domain of the 30S subunit that break multiple inter-subunit bridges. Effect of HflXr binding to the 70S ribosome shown as difference vectors for P and Cα atoms of the 30S platform domain, colored by distance. The displacement vectors are drawn between the empty and HflXr-bound head-swiveled states, structures I-B and II-C (A), classic non-rotated states bound to HPF, structures I-A and II-B (B), and the ratcheted states, structures II-A and II-D (C), respectively, superposed on the 30S subunit of the starting structures I-B, I-A and II-A (gray ribbon). The buried surface area between subunits was determined by PISA for each 70S ribosome state, non-rotated with the 30S head domain swiveled I-B and II-C (D), classic non-rotated with HPF I-A and II-B (E), and ratcheted II-A and II-D (F). (G–K) Inter-subunit bridges of the 30S subunit platform domain (B2b, B2c, B4, B7a, B7b) are disrupted in II-C (white 50S and yellow 30S) through displacement of 16S rRNA helices and ribosomal proteins from their position in I-B (gray) due to large-scale conformational changes in the platform domain of the 30S subunit caused by HflXr. Putative hydrogen bonds are indicated with dashed gray (I-B) and green (II-C) lines.

Figure 4.

Comparison of the direction of movement of the 30S platform in canonical ribosome ratcheting and HflXr-mediated recycling. (A) The surface overview of the 70S ribosome is shown with the 50S subunit colored in light blue, the body of the 30S subunit in light yellow, the platform domain of the 30S subunit in light brown, and the 30S head in yellow orange. Magnified view of the 70S ribosome is indicated in the box, and includes a part of the 50S subunit (light blue) and 16S rRNA cartoon or surface colored according to the scheme in the overview figure (A–C). Difference vectors drawn between P atoms of the 30S platform of structure I-B and HflXr-bound structure II-C, represented as orange vectors (A and C), show the orthogonal movement of the 30S platform away from the 50S subunit. Difference vectors for the same atoms drawn between structures I-B (non-rotated) and II-A (rotated) in blue gray (B and C), show the classic ratcheting movement of the 30S subunit along the 50S subunit, which is nearly perpendicular to the HflXr-mediated movement of the 30S platform (C). Labeled black arrows denote the overall direction of the movements.

Figure 5.

HflXr alters the conformation of H69 post-splitting whereas the NTD-II acts as a sensor of the PTC occupancy. (A) Rotation of HflXr in the 50S subunit (III; purple) by ∼5^o relative to its position in the 70S ribosome (II-C; pink), resulting in a displacement of the NTD, G domain and CTD of ∼3 Å. (B) Displacement of the HflXr NTD toward H69 and dissociation of the 30S subunit result in the continued movement of H69 away from the 50S subunit. (C) Residue Arg149 of the HflXr NTD-II stacks with conserved 23S rRNA nucleotides in the PTC. (D) The NTD-II and Arg149 remain anchored in the PTC in both the 70S ribosome II-C (pink) and 50S subunit III (purple) while not altering the conformation of the PTC. The tip of the PTC-binding loop of HflXr is not compatible with the lincosamide antibiotic lincomycin (turquoise, PDB 8A5I) (10). (E) NTD-I and NTD-II of HflXr are not compatible with a tRNA bound in the classic p/P state.

Figure 6.

Model of HflXr-mediated ribosome recycling. (A) After peptide release, the ribosome spontaneously samples the non-rotated and rotated states. (B) HflXr•GTP binds to the post-release rotated ribosome. (C) Upon back rotation of the 30S subunit, the NTD-I of HflXr impedes helix H69 from returning to its original position, effectively disengaging inter-subunit bridges between the platform domain of the 30S subunit and the 50S subunit. (D) Splitting of the ribosome frees tRNA whereas the dissociated 50S subunit remains bound to HflXr. (E) Following GTP hydrolysis, HflXr•GDP dissociates from the 50S subunit.