RF3-induced subunit rotation destabilizes RF1 binding. a Cryo-EM map of SSU (light blue) and RF1 (orange) from state III compared with SSU (dark blue) and RF1 (red) from state IV. b Isolated cryo-EM electron densities (grey mesh) with molecular models for RF1 from state III (orange) and state IV (red) shown at the same contour level based on comparison with the SSU density. c Domain 2/4 of RF1 from state III (sIII:RF1, orange) is rotated by 6° and shifted by 4 Å compared to RF1 from state IV (sIV:RF1, red). d, e Contacts (arrowed) between RF1 (orange) and P/P-tRNA (green) are lost upon formation of the hybrid P/E-tRNA (light blue). Amino acids of RF1 that contact P/P-tRNA are shown as spheres. e Zoom of d showing the presence or absence of RF1 contacts with the ASL of P/P- or P/E-tRNA, respectively. 

Daniel N. Wilson Research Group

Significance

During translation termination in bacteria, the release factors RF1 and RF2 are recycled from the ribosome by RF3. While high-resolution structures of the individual termination factors on the ribosome exist, direct structural insight into how RF3 mediates dissociation of the decoding RFs has been lacking. Here we have used the Apidaecin 137 peptide to trap RF1 together with RF3 on the ribosome and visualize an ensemble of termination intermediates using cryo-electron microscopy. Binding of RF3 to the ribosome induces small subunit (SSU) rotation and swivelling of the head, yielding intermediate states with shifted P-site tRNAs and RF1 conformations. RF3 does not directly eject RF1 from the ribosome, but rather induces full rotation of the SSU that indirectly dislodges RF1 from its binding site. SSU rotation is coupled to the accommodation of the GTPase domain of RF3 on the large subunit (LSU), thereby promoting GTP hydrolysis and dissociation of RF3 from the ribosome. 

Graf, M., Huter, P., Maracci, C.,  Peterek, M., Rodnina, M.V.,  and Wilson D.N.: Visualization of translation termination intermediates trapped by the Apidaecin 137 peptide during RF3-mediated recycling of RF1 Nat. Commun. (2018)9, 3053 doi:10.1038/s41467-018-05465-1

SAXS‐based structural modeling of the proC2:14‐3‐3ζ complex. (A) Best‐scoring AllosMod‐FoXS model of the proC2:14‐3‐3ζ complex shown in two perpendicular views. The N‐terminal linker, the p19 and the p12 domains and phosphorylation sites are indicated in brown, salmon, yellow, and red spheres, respectively. The protomers of 14‐3‐3ζ are shown in blue. 14‐3‐3 helices are identified with capital letters, whereas proC2 helices and β‐strands are identified with Greek letters. (B) Intermolecular cross‐links connecting the NLS region of proC2 (Lys¹⁵³) to helices H1 and H3 of 14‐3‐3 (Lys¹¹ and Lys⁶⁸); and the proC2 domain p12 (Lys³⁷², Lys³⁸¹) to the 14‐3‐3ζ helix H3 (Lys⁶⁸). Lysine residues of proC2 are shown in brown. (C) Cross‐links between the N terminus of proC2 (Ser¹²³) and the 14‐3‐3ζ helix H4 and H5/H6 loop (Lys⁷⁵, Lys¹³⁸). Lysine residues of proC2 are shown in brown.

Tomáš Obšil Research Group

Significance

The main goal of this work is to provide the structural basis for the role of 14‐3‐3 protein binding in regulating caspase‐2 activation. Because all our previous attempts to crystallize the complex between Ser139‐ and Ser164‐ phosphorylated caspase‐2 (residues 123–452 without the CARD domain, hereafter referred to as proC2) and 14‐3‐3ζ had been unsuccessful, we decided to use small angle X‐ray scattering (SAXS) combined with NMR, with chemical cross‐linking coupled to MS and with fluorescence spectroscopy to characterize the solution structure and conformational behavior of this complex.

The structural analysis of the 14‐3‐3:caspase‐2 complex reported in this study suggested that 14‐3‐3 protein binding may inhibit caspase‐2 activation through interference with caspase‐2 oligomerization and/or its nuclear localization by sterically occluding caspase‐2 p12 domain as well as NLS, which is bordered by the two phosphorylated 14‐3‐3‐binding motifs of caspase‐2. Thus, these results corroborate the hypothesis that 14‐3‐3 binding is an important regulatory element of caspase‐2 activation. Further research should be directed to study the effect of 14‐3‐3 on the caspase‐2 dimerization and cellular localization in vivo.

Smidova, A; Alblova, M.;Kalabova, D.; Psenakova, K.; Rosulek, M; Herman, P.; Obsil, T., and Obsilova, V.: 14-3-3 protein masks the nuclear localization sequence of caspase-2. Febs Journal 285, 4196-4213, doi:10.1111/febs.14670 (2018).