Implication of Stm1 in the protection of eIF5A, eEF2 and tRNA through dormant ribosomes.

Background: Dormant ribosomes are typically associated with preservation factors to protect themselves from degradation under stress conditions. Stm1/SERBP1 is one such protein that anchors the 40S and 60S subunits together. Several proteins and tRNAs bind to this complex as well, yet the molecular mechanisms remain unclear. Methods: Here, we reported the cryo-EM structures of five newly identified Stm1/SERBP1-bound ribosomes. Results: These structures highlighted that eIF5A, eEF2, and tRNA might bind to dormant ribosomes under stress to avoid their own degradation, thus facilitating protein synthesis upon the restoration of growth conditions. In addition, Ribo-seq data analysis reflected the upregulation of nutrient, metabolism, and external-stimulus-related pathways in the ∆stm1 strain, suggesting possible regulatory roles of Stm1. Discussion: The knowledge generated from the present work will facilitate in better understanding the molecular mechanism of dormant ribosomes.

LLPS of FXR proteins drives replication organelle clustering for ß-coronaviral proliferation.

ß-Coronaviruses remodel host endomembranes to form double-membrane vesicles (DMVs) as replication organelles (ROs) that provide a shielded microenvironment for viral RNA synthesis in infected cells. DMVs are clustered, but the molecular underpinnings and pathophysiological functions remain unknown. Here, we reveal that host fragile X-related (FXR) family proteins (FXR1/FXR2/FMR1) are required for DMV clustering induced by expression of viral non-structural proteins (Nsps) Nsp3 and Nsp4. Depleting FXRs results in DMV dispersion in the cytoplasm. FXR1/2 and FMR1 are recruited to DMV sites via specific interaction with Nsp3. FXRs form condensates driven by liquid-liquid phase separation, which is required for DMV clustering. FXR1 liquid droplets concentrate Nsp3 and Nsp3-decorated liposomes in vitro. FXR droplets facilitate recruitment of translation machinery for efficient translation surrounding DMVs. In cells depleted of FXRs, SARS-CoV-2 replication is significantly attenuated. Thus, SARS-CoV-2 exploits host FXR proteins to cluster viral DMVs via phase separation for efficient viral replication.

Structural Insights into the Distortion of the Ribosomal Small Subunit at Different Magnesium Concentrations.

Magnesium ions are abundant and play indispensable functions in the ribosome. A decrease in Mg2+ concentration causes 70S ribosome dissociation and subsequent unfolding. Structural distortion at low Mg2+ concentrations has been observed in an immature pre50S, while the structural changes in mature subunits have not yet been studied. Here, we purified the 30S subunits of E. coli cells under various Mg2+ concentrations and analyzed their structural distortion by cryo-electron microscopy. Upon systematically interrogating the structural heterogeneity within the 1 mM Mg2+ dataset, we observed 30S particles with different levels of structural distortion in the decoding center, h17, and the 30S head. Our model showed that, when the Mg2+ concentration decreases, the decoding center distorts, starting from h44 and followed by the shifting of h18 and h27, as well as the dissociation of ribosomal protein S12. Mg2+ deficiency also eliminates the interactions between h17, h10, h15, and S16, resulting in the movement of h17 towards the tip of h6. More flexible structures were observed in the 30S head and platform, showing high variability in these regions. In summary, the structures resolved here showed several prominent distortion events in the decoding center and h17. The requirement for Mg2+ in ribosomes suggests that the conformational changes reported here are likely shared due to a lack of cellular Mg2+ in all domains of life.

Cargo Recognition Mechanisms of Yeast Myo2 Revealed by AlphaFold2-Powered Protein Complex Prediction.

Myo2, a yeast class V myosin, transports a broad range of organelles and plays important roles in various cellular processes, including cell division in budding yeast. Despite the fact that several structures of Myo2/cargo adaptor complexes have been determined, the understanding of the versatile cargo-binding modes of Myo2 is still very limited, given the large number of cargo adaptors identified for Myo2. Here, we used ColabFold, an AlphaFold2-powered and easy-to-use tool, to predict the complex structures of Myo2-GTD and its several cargo adaptors. After benchmarking the prediction strategy with three Myo2/cargo adaptor complexes that have been determined previously, we successfully predicted the atomic structures of Myo2-GTD in complex with another three cargo adaptors, Vac17, Kar9 and Pea2, which were confirmed by our biochemical characterizations. By systematically comparing the interaction details of the six complexes of Myo2 and its cargo adaptors, we summarized the cargo-binding modes on the three conserved sites of Myo2-GTD, providing an overall picture of the versatile cargo-recognition mechanisms of Myo2. In addition, our study demonstrates an efficient and effective solution to study protein-protein interactions in the future via the AlphaFold2-powered prediction.

Sequential rescue and repair of stalled and damaged ribosome by bacterial PrfH and RtcB.

RtcB is involved in transfer RNA (tRNA) splicing in archaeal and eukaryotic organisms. However, most RtcBs are found in bacteria, whose tRNAs have no introns. Because tRNAs are the substrates of archaeal and eukaryotic RtcB, it is assumed that bacterial RtcBs are for repair of damaged tRNAs. Here, we show that a subset of bacterial RtcB, denoted RtcB2 herein, specifically repair ribosomal damage in the decoding center. To access the damage site for repair, however, the damaged 70S ribosome needs to be dismantled first, and this is accomplished by bacterial PrfH. Peptide-release assays revealed that PrfH is only active with the damaged 70S ribosome but not with the intact one. A 2.55-Å cryo-electron microscopy structure of PrfH in complex with the damaged 70S ribosome provides molecular insight into PrfH discriminating between the damaged and the intact ribosomes via specific recognition of the cleaved 3'-terminal nucleotide. RNA repair assays demonstrated that RtcB2 efficiently repairs the damaged 30S ribosomal subunit but not the damaged tRNAs. Cell-based assays showed that the RtcB2-PrfH pair reverse the damage inflicted by ribosome-specific ribotoxins in vivo. Thus, our combined biochemical, structural, and cell-based studies have uncovered a bacterial defense system specifically evolved to reverse the lethal ribosomal damage in the decoding center for cell survival.

Conserved heterodimeric GTPase Rbg1/Tma46 promotes efficient translation in eukaryotic cells.

Conserved developmentally regulated guanosine triphosphate (GTP)-binding proteins (Drgs) and their binding partner Drg family regulatory proteins (Dfrps) are important for embryonic development, cellular growth control, differentiation, and proliferation. Here, we report that the yeast Drg1/Dfrp1 ortholog Rbg1/Tma46 facilitates translational initiation, elongation, and termination by suppressing prolonged ribosome pausing. Consistent with the genome-wide observations, deletion of Rbg1 exacerbates the growth defect resulting from translation stalling, and Rbg1 stabilizes mRNAs against no-go decay. Furthermore, we provide a cryoelectron microscopy (cryo-EM) structure of the 80S ribosome bound with Rbg1/Tma46 that reveals the molecular interactions responsible for Rbg1/Tma46 function. The Rbg1 subunit binds to the GTPase association center of the ribosome and the A-tRNA, and the N-terminal zinc finger domain of the Tma46 subunit binds to the 40S, establishing an interaction critical for the ribosomal association. Our results answer the fundamental question of how a paused ribosome resumes translation and show that Drg1/Dfrp1 play a critical role in ensuring orderly translation.

Conformation of methylated GGQ in the Peptidyl Transferase Center during Translation Termination.

The universally conserved Gly-Gly-Gln (GGQ) tripeptide in release factors or release factor-like surveillance proteins is required to catalyze the release of nascent peptide in the ribosome. The glutamine of the GGQ is methylated post-translationally at the N5 position in vivo; this covalent modification is essential for optimal cell growth and efficient translation termination. However, the precise conformation of the methylated-GGQ tripeptide in the ribosome remains unknown. Using cryoEM and X-ray crystallography, we report the conformation of methylated-GGQ in the peptidyl transferase center of the ribosome during canonical translational termination and co-translation quality control. It has been suggested that the GGQ motif arose independently through convergent evolution among otherwise unrelated proteins that catalyze peptide release. The requirement for this tripeptide in the highly conserved peptidyl transferase center suggests that the conformation reported here is likely shared during termination of protein synthesis in all domains of life.

Sbp1 modulates the translation of Pab1 mRNA in a poly(A)- and RGG-dependent manner.

RNA-binding protein Sbp1 facilitates the decapping pathway in mRNA metabolism and inhibits global mRNA translation by an unclear mechanism. Here we report molecular interactions responsible for Sbp1-mediated translation inhibition of mRNA encoding the polyadenosine-binding protein (Pab1), an essential translation factor that stimulates mRNA translation and inhibits mRNA decapping in eukaryotic cells. We demonstrate that the two distal RRMs of Sbp1 bind to the poly(A) sequence in the 5'UTR of the Pab1 mRNA specifically and cooperatively while the central RGG domain of the protein interacts directly with Pab1. Furthermore, methylation of arginines in the RGG domain abolishes the protein-protein interaction and the inhibitory effect of Sbp1 on translation initiation of Pab1 mRNA. Based on these results, the underlying mechanism for Sbp1-specific translational regulation is proposed. The functional differences of Sbp1 and RGG repeats alone on transcript-specific translation were observed, and a comparison of the results suggests the importance of remodeling the 5'UTR by RNA-binding proteins in mRNA translation.

Brd4 modulates the innate immune response through Mnk2-eIF4E pathway-dependent translational control of IκBα.

Bromodomain-containing factor Brd4 has emerged as an important transcriptional regulator of NF-κB-dependent inflammatory gene expression. However, the in vivo physiological function of Brd4 in the inflammatory response remains poorly defined. We now demonstrate that mice deficient for Brd4 in myeloid-lineage cells are resistant to LPS-induced sepsis but are more susceptible to bacterial infection. Gene-expression microarray analysis of bone marrow-derived macrophages (BMDMs) reveals that deletion of Brd4 decreases the expression of a significant amount of LPS-induced inflammatory genes while reversing the expression of a small subset of LPS-suppressed genes, including MAP kinase-interacting serine/threonine-protein kinase 2 (Mknk2). Brd4-deficient BMDMs display enhanced Mnk2 expression and the corresponding eukaryotic translation initiation factor 4E (eIF4E) activation after LPS stimulation, leading to an increased translation of IκBα mRNA in polysomes. The enhanced newly synthesized IκBα reduced the binding of NF-κB to the promoters of inflammatory genes, resulting in reduced inflammatory gene expression and cytokine production. By modulating the translation of IκBα via the Mnk2-eIF4E pathway, Brd4 provides an additional layer of control for NF-κB-dependent inflammatory gene expression and inflammatory response.

Structural basis of co-translational quality control by ArfA and RF2 bound to ribosome.

Quality control mechanisms intervene appropriately when defective translation events occur, in order to preserve the integrity of protein synthesis. Rescue of ribosomes translating on messenger RNAs that lack stop codons is one of the co-translational quality control pathways. In many bacteria, ArfA recognizes stalled ribosomes and recruits the release factor RF2, which catalyses the termination of protein synthesis. Although an induced-fit mechanism of nonstop mRNA surveillance mediated by ArfA and RF2 has been reported, the molecular interaction between ArfA and RF2 in the ribosome that is responsible for the mechanism is unknown. Here we report an electron cryo-microscopy structure of ArfA and RF2 in complex with the 70S ribosome bound to a nonstop mRNA. The structure, which is consistent with our kinetic and biochemical data, reveals the molecular interactions that enable ArfA to specifically recruit RF2, not RF1, into the ribosome and to enable RF2 to release the truncated protein product in this co-translational quality control pathway. The positively charged C-terminal domain of ArfA anchors in the mRNA entry channel of the ribosome. Furthermore, binding of ArfA and RF2 induces conformational changes in the ribosomal decoding centre that are similar to those seen in other protein-involved decoding processes. Specific interactions between residues in the N-terminal domain of ArfA and RF2 help RF2 to adopt a catalytically competent conformation for peptide release. Our findings provide a framework for understanding recognition of the translational state of the ribosome by new proteins, and expand our knowledge of the decoding potential of the ribosome.

Peptide release promoted by methylated RF2 and ArfA in nonstop translation is achieved by an induced-fit mechanism.

Here we report that the specificity of peptide release in the ribosome on a nonstop mRNA by ArfA and RF2 is achieved by an induced-fit mechanism. Using RF2 that is methylated on the glutamine of its GGQ motif (RF2(m)), we show that methylation substantially increases the rate of ArfA/RF2-catalyzed peptide release on a nonstop mRNA that does not occupy the ribosomal A site, but has only a modest effect on k(cat) by the same proteins on longer nonstop mRNAs occupying the A site of the mRNA channel in the ribosome. Our data suggest that enhancement in the kcat of peptide release by ArfA and RF2 under the cognate decoding condition is the result of favorable conformational changes in the nonstop complex. We demonstrate a shared mechanism between canonical and nonstop termination, supported by similarities in the kinetic mechanisms in antibiotic inhibition and methylation-correlated enhancement in the rate of peptide release. Despite these similarities, our data suggest that nonstop termination differs from canonical pathway in the downstream event of recycling.

Crystal Structure of Human SSRP1 Middle Domain Reveals a Role in DNA Binding.

SSRP1 is a subunit of the FACT complex, an important histone chaperone required for transcriptional regulation, DNA replication and damage repair. SSRP1 also plays important roles in transcriptional regulation independent of Spt16 and interacts with other proteins. Here, we report the crystal structure of the middle domain of SSRP1. It consists of tandem pleckstrin homology (PH) domains. These domains differ from the typical PH domain in that PH1 domain has an extra conserved ßαß topology. SSRP1 contains the well-characterized DNA-binding HMG-1 domain. Our studies revealed that SSRP1-M can also participate in DNA binding, and that this binding involves one positively charged patch on the surface of the structure. In addition, SSRP1-M did not bind to histones, which was assessed through pull-down assays. This aspect makes the protein different from other related proteins adopting the double PH domain structure. Our studies facilitate the understanding of SSRP1 and provide insights into the molecular mechanisms of interaction with DNA and histones of the FACT complex.

Crystal structure of phospholipase PA2-Vb, a protease-activated receptor agonist from the Trimeresurus stejnegeri snake venom.

Phospholipase A2 (PLA2) is an important component in snake venoms. Here, an acidic PLA2, designated PA2-Vb was isolated from the Trimeresurus stejnegeri snake venom. PA2-Vb acts on a protease-activated receptor (PAR-1) to evoke Ca(2+) release through the inositol 1,4,5-trisphosphate receptor (IP3R) and induces mouse aorta contraction. PAR-1, phospholipase C and IP3R inhibitors suppressed PA2-Vb-induced aorta contraction. The crystal structure reveals that PA2-Vb has the typical fold of most snake venom PLA2. Several PEG molecules bond to a positively charged pocket. The finding offers a novel pharmacological basis of the structure for investigating the PAR-1 receptor and suggests potential applications for PA2-Vb in the vascular system.

Crystal structures and RNA-binding properties of the RNA recognition motifs of heterogeneous nuclear ribonucleoprotein L: insights into its roles in alternative splicing regulation.

Heterogeneous nuclear ribonucleoprotein L (hnRNP L) is an abundant RNA-binding protein implicated in many bioprocesses, including pre-mRNA processing, mRNA export of intronless genes, internal ribosomal entry site-mediated translation, and chromatin modification. It contains four RNA recognition motifs (RRMs) that bind with CA repeats or CA-rich elements. In this study, surface plasmon resonance spectroscopy assays revealed that all four RRM domains contribute to RNA binding. Furthermore, we elucidated the crystal structures of hnRNP L RRM1 and RRM34 at 2.0 and 1.8 Å, respectively. These RRMs all adopt the typical ß1α1ß2ß3α2ß4 topology, except for an unusual fifth ß-strand in RRM3. RRM3 and RRM4 interact intimately with each other mainly through helical surfaces, leading the two ß-sheets to face opposite directions. Structure-based mutations and surface plasmon resonance assay results suggested that the ß-sheets of RRM1 and RRM34 are accessible for RNA binding. FRET-based gel shift assays (FRET-EMSA) and steady-state FRET assays, together with cross-linking and dynamic light scattering assays, demonstrated that hnRNP L RRM34 facilitates RNA looping when binding to two appropriately separated binding sites within the same target pre-mRNA. EMSA and isothermal titration calorimetry binding studies with in vivo target RNA suggested that hnRNP L-mediated RNA looping may occur in vivo. Our study provides a mechanistic explanation for the dual functions of hnRNP L in alternative splicing regulation as an activator or repressor.

AhV_aPA-induced vasoconstriction involves the IP3Rs-mediated Ca²âº releasing.

AhV_aPA, the acidic PLA2 purified from Agkistrodon halys pallas venom, was previously reported to possess a strong enzymatic activity and can remarkably induce a further contractile response on the 60 mM K⁺-induced contraction with an EC50 in 369 nM on mouse thoracic aorta rings. In the present study, we found that the p-bromo-phenacyl-bromide (pBPB), which can completely inhibit the enzymatic activity of AhV_aPA, did not significantly reduce the contractile response on vessel rings induced by AhV_aPA, indicating that the vasoconstrictor effects of AhV_aPA are independent of the enzymatic activity. The inhibitor experiments showed that the contractile response induced by AhV_aPA is mainly attributed to the Ca²âº releasing from Ca²âº store, especially sarcoplasmic reticulum (SR). Detailed studies showed that the Ca²âº release from SR is related to the activation of inositol trisphosphate receptors (IP3Rs) rather than ryanodine receptors (RyRs). Furthermore, the vasoconstrictor effect could be strongly reduced by pre-incubation with heparin, indicating that the basic amino acid residues on the surface of AhV_aPA may be involved in the interaction between AhV_aPA and the molecular receptors. These findings offer new insights into the functions of snake PLA2 and provide a novel pathogenesis of A. halys pallas venom.

The structure of the ARE-binding domains of Hu antigen R (HuR) undergoes conformational changes during RNA binding.

Human RNA-binding protein (HuR), a ubiquitously expressed member of the Hu protein family, plays an important role in mRNA degradation and has been implicated as a key post-transcriptional regulator. HuR contains three RNA-recognition motif (RRM) domains. The two N-terminal tandem RRM domains can selectively bind AU-rich elements (AREs), while the third RRM domain (RRM3) contributes to interactions with the poly-A tail of target mRNA and other ligands. Here, the X-ray structure of two methylated tandem RRM domains (RRM1/2) of HuR in their RNA-free form was solved at 2.9 Šresolution. The crystal structure of RRM1/2 complexed with target mRNA was also solved at 2.0 Šresolution; comparisons of the two structures show that HuR RRM1/2 undergoes conformational changes upon RNA binding. Fluorescence polarization assays (FPA) were used to study the protein-RNA interactions. Both the structure and the FPA analysis indicated that RRM1 is the primary ARE-binding domain in HuR and that the conformational changes induce subsequent contacts of the RNA substrate with the inter-domain linker and RRM2 which greatly improve the RNA-binding affinity of HuR.

Crystal structure and activating effect on RyRs of AhV_TL-I, a glycosylated thrombin-like enzyme from Agkistrodon halys snake venom.

A snake venom thrombin-like enzyme (SVTLE) from Agkistrodon halys pallas venom was isolated by means of a two-step chromatographic procedure. The purified enzyme, named AhV_TL-I, showed fibrinogenolytic activity against both the Aα and Bß chains of bovine fibrinogen. Unlike the other SVTLEs, AhV_TL-I has poor esterolytic activity upon BAEE substrate. The N-terminal sequence of AhV_TL-I was determined to be IIGGDEXNINEHRFLVALYT, and the molecular mass was confirmed to 29389.533 Da by MALDI-TOF mass spectrometry. Its complete cDNA and derived amino acid sequence were obtained by RT-PCR. The crystal structure of AhV_TL-I was determined at a resolution of 1.75 Å. A disaccharide was clearly mapped in the structure, which involved in regulating the esterolytic activity of AhV_TL-I. The presence of the N-glycan deformed the 99-loop, and the resulting steric hindrances hindered the substrates to access the active site. Furthermore, with the carbohydrate moiety, AhV_TL-I could induce mouse thoracic aortic ring contraction with the EC(50) of 147 nmol/L. Besides, the vasoconstrictor effects of AhV_TL-I were also independent of the enzymatic activity. The results of [Ca(2+)](i) measurement showed that the vasoconstrictor effects of AhV_TL-I were attributed to Ca(2+) releasing from Ca(2+) store. Further studies showed that it was related to the activation of ryanodine receptors (RyRs). These offer new insights into the snake SVTLEs functions and provide a novel pathogenesis of A. halys pallas venom.

Purification, crystallization and preliminary X-ray diffraction analysis of an acidic phospholipase A2 with vasoconstrictor activity from Agkistrodon halys pallas venom.

Phospholipases A2 (PLA2s) are the major component of snake venoms and exert a variety of relevant toxic actions such as neurotoxicity and myotoxicity, amongst others. An acidic PLA2, here named AhV_aPA, was purified from Agkistrodon halys pallas venom by means of a three-step chromatographic procedure. AhV_aPA migrated as a single band on SDS-PAGE gels, with a molecular weight of about 14 kDa. Like other acidic aPLA2s, AhV_aPA has high enzymatic activity. Tension measurements of mouse thoracic aortic rings remarkably indicated that AhV_aPA could induce a further contractile response on the 60 mM K+-induced contraction, with an EC50 of 369 nmol l(-1). Rod-shaped crystals were obtained by the hanging-drop vapour-diffusion method and diffracted to a resolution limit of 2.30 Å. The crystals belonged to space group P222, with unit-cell parameters a=44.27, b=68.39, c=81.54 Å.

Crystallization and preliminary crystallographic studies of UbiG, an O-methyltransferase from Escherichia coli.

UbiG, an O-methyltransferase from the ubiquinone-biosynthesis pathway in Escherichia coli, catalyzes two O-methyl transfer steps. The primary structures of the O-methyltransferase enzyme family used in ubiquinone synthesis are conserved in both prokaryotes and eukaryotes, but their tertiary structures and catalytic mechanisms are not yet known. Here, UbiG with an N-terminal hexahistidine tag was expressed and crystallized. Crystals grown by the hanging-drop vapour-diffusion method diffracted to 2.00 Šresolution and belonged to space group C121, with unit-cell parameters a = 119.8, b = 58.6, c = 40.2 Å, ß = 105.3°. Both Matthews coefficient analysis and the self-rotation function suggested the presence of one molecule per asymmetric unit in the crystal, with a solvent content of 50.52% (V(M) = 2.48 Å(3) Da(-1)).

Semi-synthesis and biological activity of gamma-lactones analogs of camptothecin.

A series of E-ring gamma-lactone camptothecin derivatives were synthesized by semi-synthesis via a three-step domino reaction. Their biological activity was evaluated on two types of human tumor cell lines A549 and HT-29 with sulforhodamine-B (SRB) method. The antitumor activity of these compounds was lower than SN-38, only compound 12c was found to be close to the activity of Topotecan. The structure-activity relationship (SAR) of these analogs was studied and discussed.