Cotranslational protein folding depends on general chaperones that engage highly diverse nascent chains at the ribosomes. Here we discover a dedicated ribosome-associated chaperone, Chp1, that ...rewires the cotranslational folding machinery to assist in the challenging biogenesis of abundantly expressed eukaryotic translation elongation factor 1A (eEF1A). Our results indicate that during eEF1A synthesis, Chp1 is recruited to the ribosome with the help of the nascent polypeptide-associated complex (NAC), where it safeguards eEF1A biogenesis. Aberrant eEF1A production in the absence of Chp1 triggers instant proteolysis, widespread protein aggregation, activation of Hsf1 stress transcription and compromises cellular fitness. The expression of pathogenic eEF1A2 variants linked to epileptic-dyskinetic encephalopathy is protected by Chp1. Thus, eEF1A is a difficult-to-fold protein that necessitates a biogenesis pathway starting with dedicated folding factor Chp1 at the ribosome to protect the eukaryotic cell from proteostasis collapse.
The highly conserved 90 kDa heat shock protein (Hsp90) chaperones use ATP to regulate the stability and activity of many signalling molecules like protein kinases and transcription factors. Studies ...using crystallography, electron microscopy and small‐angle X‐ray scattering yielded controversial results for the conformational states that these dimeric multidomain proteins assume while progressing through the ATPase cycle. To better understand the molecular mechanism of Hsp90 proteins, we studied the conformational dynamics of the Escherichia coli homologue HtpG in solution using amide hydrogen exchange mass spectrometry (HX‐MS) and fluorescence spectroscopy. A conformation‐sensitive fluorescent probe allowed to elucidate the ATPase cycle of HtpG. Continuous‐labelling and pulse‐labelling HX‐MS experiments revealed major ATP‐induced conformational changes throughout the protein that do not occur simultaneously, but progress surprisingly slow from the immediate nucleotide‐binding site towards the N terminus and the middle domain. The conversion between the different conformational states is rate limiting for ATP hydrolysis, and the nucleotide‐coordinating residue, Glu34, is important for the rate constant of conversion. Our findings, for the first time, allow to kinetically resolve changes in the conformational dynamics of individual structural elements of Hsp90.
Protein synthesis must be finely tuned in the developing nervous system as the final essential step of gene expression. This study investigates the architecture of ribosomes from the neocortex during ...neurogenesis, revealing Ebp1 as a high-occupancy 60S peptide tunnel exit (TE) factor during protein synthesis at near-atomic resolution by cryoelectron microscopy (cryo-EM). Ribosome profiling demonstrated Ebp1-60S binding is highest during start codon initiation and N-terminal peptide elongation, regulating ribosome occupancy of these codons. Membrane-targeting domains emerging from the 60S tunnel, which recruit SRP/Sec61 to the shared binding site, displace Ebp1. Ebp1 is particularly abundant in the early-born neural stem cell (NSC) lineage and regulates neuronal morphology. Ebp1 especially impacts the synthesis of membrane-targeted cell adhesion molecules (CAMs), measured by pulsed stable isotope labeling by amino acids in cell culture (pSILAC)/bioorthogonal noncanonical amino acid tagging (BONCAT) mass spectrometry (MS). Therefore, Ebp1 is a central component of protein synthesis, and the ribosome TE is a focal point of gene expression control in the molecular specification of neuronal morphology during development.
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•Near-atomic resolution structure of translating ribosomes in the nervous system•Ebp1 binds the 60S peptide tunnel exit during active protein synthesis•Ebp1 regulates start codon initiation and N-terminal peptide elongation•Ebp1 is abundant in early-born neocortex neurons and regulates their morphology
Kraushar et al. visualize protein synthesis in the developing mouse brain at near-atomic resolution. Ebp1 binds the 60S tunnel exit to regulate translation initiation and N-terminal peptide elongation proteome-wide. Ebp1 is particularly abundant in early-born neocortex neural stem cells and regulates neuronal morphology, impacting cell adhesion molecule synthesis.
Newly synthesized polypeptides undergo various cotranslational maturation steps, including N-terminal enzymatic processing, chaperone-assisted folding and membrane targeting, but the spatial and ...temporal coordination of these steps is unclear. We show that Escherichia coli methionine aminopeptidase (MAP) associates with ribosomes through a charged loop that is crucial for nascent-chain processing and cell viability. MAP competes with peptide deformylase (PDF), the first enzyme to act on nascent chains, for binding sites at the ribosomal tunnel exit. PDF has extremely fast association and dissociation kinetics, which allows it to frequently sample ribosomes and ensure the processing of nascent chains after their emergence. Premature recruitment of the chaperone trigger factor, or polypeptide folding, negatively affect processing efficiency. Thus, the fast ribosome association kinetics of PDF and MAP are crucial for the temporal separation of nascent-chain processing from later maturation events, including chaperone recruitment and folding.
Growing cells invest a significant part of their biosynthetic capacity into the production of proteins. To become functional, newly-synthesized proteins must be N-terminally processed, folded and ...often translocated to other cellular compartments. A general strategy is to integrate these protein maturation processes with translation, by cotranslationally engaging processing enzymes, chaperones and targeting factors with the nascent polypeptide. Precise coordination of all factors involved is critical for the efficiency and accuracy of protein synthesis and cellular homeostasis. This review provides an overview of the current knowledge on cotranslational protein maturation, with a focus on the production of cytosolic proteins in bacteria. We describe the role of the ribosome and the chaperone network in protein folding and how the dynamic interplay of all cotranslationally acting factors guides the sequence of cotranslational events. Finally, we discuss recent data demonstrating the coupling of protein synthesis with the assembly of protein complexes and end with a brief discussion of outstanding questions and emerging concepts in the field of cotranslational protein maturation.
The rates at which domains fold and codons are translated are important factors in determining whether a nascent protein will co-translationally fold and function or misfold and malfunction. Here we ...develop a chemical kinetic model that calculates a protein domain's co-translational folding curve during synthesis using only the domain's bulk folding and unfolding rates and codon translation rates. We show that this model accurately predicts the course of co-translational folding measured in vivo for four different protein molecules. We then make predictions for a number of different proteins in yeast and find that synonymous codon substitutions, which change translation-elongation rates, can switch some protein domains from folding post-translationally to folding co-translationally--a result consistent with previous experimental studies. Our approach explains essential features of co-translational folding curves and predicts how varying the translation rate at different codon positions along a transcript's coding sequence affects this self-assembly process.
Incorporating fluorescent amino acids by suppression of the TAG amber codon is a useful tool for site-specific labeling of proteins and visualizing their localization in living cells. Here we use a ...plasmid encoded orthogonal tRNA/aminoacyl-tRNA synthetase pair to site-specifically label firefly luciferase with the environmentally sensitive fluorescent amino acid, 3-(6-acetylnaphthalen-2-ylamino)-2- aminopropanoic acid (ANAP) and explore the detectability of conformational changes in labeled luciferase in the yeast cytoplasm. We find that ANAP labeling efficiency is greatly increased in PSI+ cells and show that analysis of the ANAP fluorescence emission by confocal imaging allows for tracking the thermal unfolding and aggregation of luciferase in vivo. Furthermore we demonstrate that flow cytometry can be used to study conformational changes in luciferase and chaperone-mediated refolding in quantitative terms and at the level of single cells. This experimental setup for the first time allows for the direct analysis of the folding state of a protein in living cells and may serve as valuable new tool for examining mechanisms of protein folding, misfolding and aggregation.
The chaperone heat shock protein 90 (Hsp90) is well known to undergo important conformational changes, which depend on nucleotide and substrate interactions. Conversely, how the conformations of its ...unstable and disordered substrates are affected by Hsp90 is difficult to address experimentally yet is central to its function. Here, using optical tweezers, we find that Hsp90 promotes local contractions in unfolded chains that drive their global compaction down to dimensions of folded states. This compaction has a gradual nature while showing small steps, is stimulated by ATP, and performs mechanical work against counteracting forces that expand the chain dimensions. The Hsp90 interactions suppress the formation of larger-scale folded, misfolded, and aggregated structures. The observations support a model in which Hsp90 alters client conformations directly by promoting local intra-chain interactions while suppressing distant ones. We conjecture that chain compaction may be central to how Hsp90 protects unstable clients and cooperates with Hsp70.
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•The Hsp90 chaperone can actively compact its client protein chains•This induced compaction depends on ATP and performs mechanical work•The Hsp90 interactions can suppress the formation of misfolded and aggregated structures
By mechanically tethering individual protein clients at their termini with optical tweezers, Mashaghi et al. show that the chaperone Hsp90 can actively compact its unfolded substrates. This collapse-like process may have important consequences for folding and interactions with the Hsp70 system.
The molecular chaperone Hsp90 is essential in eukaryotes, in which it facilitates the folding of developmental regulators and signal transduction proteins known as Hsp90 clients. In contrast, Hsp90 ...is not essential in bacteria, and a broad characterization of its molecular and organismal function is lacking. To enable such characterization, we used a genome-scale phylogenetic analysis to identify genes that co-evolve with bacterial Hsp90. We find that genes whose gain and loss were coordinated with Hsp90 throughout bacterial evolution tended to function in flagellar assembly, chemotaxis, and bacterial secretion, suggesting that Hsp90 may aid assembly of protein complexes. To add to the limited set of known bacterial Hsp90 clients, we further developed a statistical method to predict putative clients. We validated our predictions by demonstrating that the flagellar protein FliN and the chemotaxis kinase CheA behaved as Hsp90 clients in Escherichia coli, confirming the predicted role of Hsp90 in chemotaxis and flagellar assembly. Furthermore, normal Hsp90 function is important for wild-type motility and/or chemotaxis in E. coli. This novel function of bacterial Hsp90 agreed with our subsequent finding that Hsp90 is associated with a preference for multiple habitats and may therefore face a complex selection regime. Taken together, our results reveal previously unknown functions of bacterial Hsp90 and open avenues for future experimental exploration by implicating Hsp90 in the assembly of membrane protein complexes and adaptation to novel environments.
During translation, the first encounter of nascent polypeptides is with the ribosome-associated chaperones that assist the folding process-a principle that seems to be conserved in evolution. In ...Escherichia coli, the ribosome-bound Trigger Factor chaperones the folding of cytosolic proteins by interacting with nascent polypeptides. Here we identify a ribosome-binding motif in the amino-terminal domain of Trigger Factor. We also show the formation of crosslinked products between Trigger Factor and two adjacent ribosomal proteins, L23 and L29, which are located at the exit of the peptide tunnel in the ribosome. L23 is essential for the growth of E. coli and the association of Trigger Factor with the ribosome, whereas L29 is dispensable in both processes. Mutation of an exposed glutamate in L23 prevents Trigger Factor from interacting with ribosomes and nascent chains, and causes protein aggregation and conditional lethality in cells that lack the protein repair function of the DnaK chaperone. Purified L23 also interacts specifically with Trigger Factor in vitro. We conclude that essential L23 provides a chaperone docking site on ribosomes that directly links protein biosynthesis with chaperone-assisted protein folding.
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