Human tumor suppressor protein Pdcd4 binds at the mRNA entry channel in the 40S small ribosomal subunit

Translation is regulated mainly in the initiation step, and its dysregulation is implicated in many human diseases. Several proteins have been found to regulate translational initiation, including Pdcd4 (programmed cell death gene 4). Pdcd4 is a tumor suppressor protein that prevents cell growth, invasion, and metastasis. It is downregulated in most tumor cells, while global translation in the cell is upregulated. To understand the mechanisms underlying translational control by Pdcd4, we used single-particle cryo-electron microscopy to determine the structure of human Pdcd4 bound to 40S small ribosomal subunit, including Pdcd4-40S and Pdcd4-40S-eIF4A-eIF3-eIF1 complexes. The structures reveal the binding site of Pdcd4 at the mRNA entry site in the 40S, where the C-terminal domain (CTD) interacts with eIF4A at the mRNA entry site, while the N-terminal domain (NTD) is inserted into the mRNA channel and decoding site. The structures, together with quantitative binding and in vitro translation assays, shed light on the critical role of the NTD for the recruitment of Pdcd4 to the ribosomal complex and suggest a model whereby Pdcd4 blocks the eIF4F-independent role of eIF4A during recruitment and scanning of the 5' UTR of mRNA

Escherichia coli L7/L12 ribosomal stalk complex - Insights from ion mobility mass spectrometry

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Structural basis for the inhibition of translation through eIF2α phosphorylation

11 páginas, 5 figuras, 1 tabla.One of the responses to stress by eukaryotic cells is the down-regulation of protein synthesis by phosphorylation of translation initiation factor eIF2. Phosphorylation results in low availability of the eIF2 ternary complex (eIF2-GTP-tRNAi) by affecting the interaction of eIF2 with its GTP-GDP exchange factor eIF2B. We have determined the cryo-EM structure of yeast eIF2B in complex with phosphorylated eIF2 at an overall resolution of 4.2 Å. Two eIF2 molecules bind opposite sides of an eIF2B hetero-decamer through eIF2α-D1, which contains the phosphorylated Ser51. eIF2α-D1 is mainly inserted between the N-terminal helix bundle domains of δ and α subunits of eIF2B. Phosphorylation of Ser51 enhances binding to eIF2B through direct interactions of phosphate groups with residues in eIF2Bα and indirectly by inducing contacts of eIF2α helix 58-63 with eIF2Bδ leading to a competition with Met-tRNAi.This study was supported by the MRC-LMB EM Facility. This work was supported by grants from the Medical Research Council (MC_U105184332) and the Wellcome Trust (WT096570) to V.R. and by a grant BFU2017-85814-P from the Spanish government to J.L.L.Peer reviewe

Subtle conformational changes of the Escherichia coli ribosomal stalk complex probed by H/D exchange ESMS

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Acetylation of L12 increases interactions in the Escherichia coli ribosomal stalk complex.

The ribosomal stalk complex in Escherichia coli consists of L10 and four copies of L7/L12, and is largely responsible for binding and recruiting translation factors. Structural characterisation of this stalk complex is difficult, primarily due to its dynamics. Here, we apply mass spectrometry to follow post-translational modifications and their effect on structural changes of the stalk proteins on intact ribosomes. Our results show that increased acetylation of L12 occurs during the stationary phase on ribosomes harvested from cells grown under optimal conditions. For cells grown in minimal medium, L12 acetylation and processing is altered, resulting in deficient removal of N-terminal methionine in approximately 50% of the L12 population, while processed L12 is almost 100% acetylated. Our results show also that N-acetylation of L12 correlates with an increased stability of the stalk complex in the gas phase. To investigate further the basis of this increased stability, we applied a solution phase hydrogen deuterium exchange protocol to compare the rate of deuterium incorporation in the proteins L9, L10, L11 and L12 as well as the acetylated form of L12 (L7), in situ on the ribosome. Results show that deuterium incorporation is consistently slower for L7 relative to L12 and for L10 when L7 is predominant. Our results imply a tightening of the interaction between L7 and L10 relative to that between L12 and L10. Since acetylation is predominant when cells are grown in minimal medium, we propose that these modifications form part of the cell's strategy to increase stability of the stalk complex under conditions of stress. More generally, our results demonstrate that it is possible to discern the influence of a 42 Da post-translational modification by mass spectrometry and to record subtle changes in hydrogen/deuterium exchange within the context of an intact 2.5 MDa particle.Journal ArticleResearch Support, Non-U.S. Gov'tinfo:eu-repo/semantics/publishe

Insight into the Escherichia coli L7/L12 ribosomal stalk complex from hydrogen exchange mass spectrometry

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Subunit Dynamics and Nucleotide-Dependent Asymmetry of an AAA(+) Transcription Complex.

International audience: Bacterial enhancer binding proteins (bEBPs) are transcription activators that belong to the AAA(+) protein family. They form higher-order self-assemblies to regulate transcription initiation at stress response and pathogenic promoters. The precise mechanism by which these ATPases utilize ATP binding and hydrolysis energy to remodel their substrates remains unclear. Here we employed mass spectrometry of intact complexes to investigate subunit dynamics and nucleotide occupancy of the AAA(+) domain of one well-studied bEBP in complex with its substrate, the σ(54) subunit of RNA polymerase. Our results demonstrate that the free AAA(+) domain undergoes significant changes in oligomeric states and nucleotide occupancy upon σ(54) binding. Such changes likely correlate with one transition state of ATP and are associated with an open spiral ring formation that is vital for asymmetric subunit function and interface communication. We confirmed that the asymmetric subunit functionality persists for open promoter complex formation using single-chain forms of bEBP lacking the full complement of intact ATP hydrolysis sites. Outcomes reconcile low- and high-resolution structures and yield a partial sequential ATP hydrolysis model for bEBPs

Molecular basis for inner kinetochore configuration through RWD domain–peptide interactions

Abstract Kinetochores are dynamic cellular structures that connect chromosomes to microtubules. They form from multi‐protein assemblies that are evolutionarily conserved between yeasts and humans. One of these assemblies—COMA—consists of subunits Ame1CENP‐U, Ctf19CENP‐P, Mcm21CENP‐O and Okp1CENP‐Q. A description of COMA molecular organization has so far been missing. We defined the subunit topology of COMA, bound with inner kinetochore proteins Nkp1 and Nkp2, from the yeast Kluyveromyces lactis, with nanoflow electrospray ionization mass spectrometry, and mapped intermolecular contacts with hydrogen‐deuterium exchange coupled to mass spectrometry. Our data suggest that the essential Okp1 subunit is a multi‐segmented nexus with distinct binding sites for Ame1, Nkp1‐Nkp2 and Ctf19‐Mcm21. Our crystal structure of the Ctf19‐Mcm21 RWD domains bound with Okp1 shows the molecular contacts of this important inner kinetochore joint. The Ctf19‐Mcm21 binding motif in Okp1 configures a branch of mitotic inner kinetochores, by tethering Ctf19‐Mcm21 and Chl4CENP‐N‐Iml3CENP‐L. Absence of this motif results in dependence on the mitotic checkpoint for viability

Large-scale movement of eIF3 domains during translation initiation modulate start codon selection

21 páginas, 7 figuras, 2 tablas. Funding for open access charge: Consejo Superior de Investigaciones Científicas/SpanishNational Research CouncilThe eukaryotic initiation factor 3 (eIF3) complex is involved in every step of translation initiation, but there is limited understanding of its molecular functions. Here, we present a single particle electron cryomicroscopy (cryo-EM) reconstruction of yeast 48S ribosomal preinitiation complex (PIC) in an open conformation conducive to scanning, with core subunit eIF3b bound on the 40S interface near the decoding center in contact with the ternary complex eIF2·GTP·initiator tRNA. eIF3b is relocated together with eIF3i from their solvent interface locations observed in other PIC structures, with eIF3i lacking 40S contacts. Re-processing of micrographs of our previous 48S PIC in a closed state also suggests relocation of the entire eIF3b-3i-3g-3a-Cter module during the course of initiation. Genetic analysis indicates that high fidelity initiation depends on eIF3b interactions at the 40S subunit interface that promote the closed PIC conformation, or facilitate the relocation of eIF3b/eIF3i to the solvent interface, on start codon selection.Spanish government [BFU2017-85814-P]; Generalitat Valenciana [SEJI/2019/030 to J.L.L.]; start-up funds [R(IV)090/1076/2017-4252] from the Indian Institute of Science, Bangalore, India; Wellcome Trust/DBT India Alliance Fellowship [IA/I/17/2/503313 to T.H.]; Human Frontiers in Science Program [RGP-0028/2009]; Intramural Research Program of the NIH (to A.G.H.); T.H. also thanks DST-FIST [SR/FST/LS11-036/2014(C)], UGCSAP [F.4.13/2018/DRS-III (SAP-II)]; DBT-IISc Partnership Program Phase-II [BT/PR27952-NF/22/212/2018] for infrastructure and financial support. Funding for open access charge: Consejo Superior de Investigaciones Cient´ıficas/SpanishNational Research Council.Peer reviewe