Bayesian reweighting of biomolecular structural ensembles using heterogeneous cryo-EM maps with the cryoENsemble method.

Cryogenic electron microscopy (cryo-EM) has emerged as a powerful method for the determination of structures of complex biological molecules. The accurate characterisation of the dynamics of such systems, however, remains a challenge. To address this problem, we introduce cryoENsemble, a method that applies Bayesian reweighting to conformational ensembles derived from molecular dynamics simulations to improve their agreement with cryo-EM data, thus enabling the extraction of dynamics information. We illustrate the use of cryoENsemble to determine the dynamics of the ribosome-bound state of the co-translational chaperone trigger factor (TF). We also show that cryoENsemble can assist with the interpretation of low-resolution, noisy or unaccounted regions of cryo-EM maps. Notably, we are able to link an unaccounted part of the cryo-EM map to the presence of another protein (methionine aminopeptidase, or MetAP), rather than to the dynamics of TF, and model its TF-bound state. Based on these results, we anticipate that cryoENsemble will find use for challenging heterogeneous cryo-EM maps for biomolecular systems encompassing dynamic components.

The ribosome lowers the entropic penalty of protein folding.

Most proteins fold during biosynthesis on the ribosome1, and co-translational folding energetics, pathways and outcomes of many proteins have been found to differ considerably from those in refolding studies2-10. The origin of this folding modulation by the ribosome has remained unknown. Here we have determined atomistic structures of the unfolded state of a model protein on and off the ribosome, which reveal that the ribosome structurally expands the unfolded nascent chain and increases its solvation, resulting in its entropic destabilization relative to the peptide chain in isolation. Quantitative 19F NMR experiments confirm that this destabilization reduces the entropic penalty of folding by up to 30 kcal mol-1 and promotes formation of partially folded intermediates on the ribosome, an observation that extends to other protein domains and is obligate for some proteins to acquire their active conformation. The thermodynamic effects also contribute to the ribosome protecting the nascent chain from mutation-induced unfolding, which suggests a crucial role of the ribosome in supporting protein evolution. By correlating nascent chain structure and dynamics to their folding energetics and post-translational outcomes, our findings establish the physical basis of the distinct thermodynamics of co-translational protein folding.

Thermodynamic profiles for cotranslational trigger factor substrate recognition.

Molecular chaperones are central to the maintenance of proteostasis in living cells. A key member of this protein family is trigger factor (TF), which acts throughout the protein life cycle and has a ubiquitous role as the first chaperone encountered by proteins during synthesis. However, our understanding of how TF achieves favorable interactions with such a diverse substrate base remains limited. Here, we use microfluidics to reveal the thermodynamic determinants of this process. We find that TF binding to empty 70S ribosomes is enthalpy-driven, with micromolar affinity, while nanomolar affinity is achieved through a favorable entropic contribution for both intrinsically disordered and folding-competent nascent chains. These findings suggest a general mechanism for cotranslational TF function, which relies on occupation of the exposed TF-substrate binding groove rather than specific complementarity between chaperone and nascent chain. These insights add to our wider understanding of how proteins can achieve broad substrate specificity.

The ribosome stabilizes partially folded intermediates of a nascent multi-domain protein.

Co-translational folding is crucial to ensure the production of biologically active proteins. The ribosome can alter the folding pathways of nascent polypeptide chains, yet a structural understanding remains largely inaccessible experimentally. We have developed site-specific labelling of nascent chains to detect and measure, using 19F nuclear magnetic resonance (NMR) spectroscopy, multiple states accessed by an immunoglobulin-like domain within a tandem repeat protein during biosynthesis. By examining ribosomes arrested at different stages during translation of this common structural motif, we observe highly broadened NMR resonances attributable to two previously unidentified intermediates, which are stably populated across a wide folding transition. Using molecular dynamics simulations and corroborated by cryo-electron microscopy, we obtain models of these partially folded states, enabling experimental verification of a ribosome-binding site that contributes to their high stabilities. We thus demonstrate a mechanism by which the ribosome could thermodynamically regulate folding and other co-translational processes.

Modulating co-translational protein folding by rational design and ribosome engineering.

Co-translational folding is a fundamental process for the efficient biosynthesis of nascent polypeptides that emerge through the ribosome exit tunnel. To understand how this process is modulated by the shape and surface of the narrow tunnel, we have rationally engineered three exit tunnel protein loops (uL22, uL23 and uL24) of the 70S ribosome by CRISPR/Cas9 gene editing, and studied the co-translational folding of an immunoglobulin-like filamin domain (FLN5). Our thermodynamics measurements employing 19F/15N/methyl-TROSY NMR spectroscopy together with cryo-EM and molecular dynamics simulations reveal how the variations in the lengths of the loops present across species exert their distinct effects on the free energy of FLN5 folding. A concerted interplay of the uL23 and uL24 loops is sufficient to alter co-translational folding energetics, which we highlight by the opposite folding outcomes resulting from their extensions. These subtle modulations occur through a combination of the steric effects relating to the shape of the tunnel, the dynamic interactions between the ribosome surface and the unfolded nascent chain, and its altered exit pathway within the vestibule. These results illustrate the role of the exit tunnel structure in co-translational folding, and provide principles for how to remodel it to elicit a desired folding outcome.

Thermodynamics of co-translational folding and ribosome-nascent chain interactions.

Proteins can begin the conformational search for their native structure in parallel with biosynthesis on the ribosome, in a process termed co-translational folding. In contrast to the reversible folding of isolated domains, as a nascent chain emerges from the ribosome exit tunnel during translation the free energy landscape it explores also evolves as a function of chain length. While this presents a substantially more complex measurement problem, this review will outline the progress that has been made recently in understanding, quantitatively, the process by which a nascent chain attains its full native stability, as well as the mechanisms through which interactions with the nearby ribosome surface can perturb or modulate this process.

Common sequence motifs of nascent chains engage the ribosome surface and trigger factor.

In the cell, the conformations of nascent polypeptide chains during translation are modulated by both the ribosome and its associated molecular chaperone, trigger factor. The specific interactions that underlie these modulations, however, are still not known in detail. Here, we combine protein engineering, in-cell and in vitro NMR spectroscopy, and molecular dynamics simulations to explore how proteins interact with the ribosome during their biosynthesis before folding occurs. Our observations of α-synuclein nascent chains in living Escherichia coli cells reveal that ribosome surface interactions dictate the dynamics of emerging disordered polypeptides in the crowded cytosol. We show that specific basic and aromatic motifs drive such interactions and directly compete with trigger factor binding while biasing the direction of the nascent chain during its exit out of the tunnel. These results reveal a structural basis for the functional role of the ribosome as a scaffold with holdase characteristics and explain how handover of the nascent chain to specific auxiliary proteins occurs among a host of other factors in the cytosol.

Nascent chains can form co-translational folding intermediates that promote post-translational folding outcomes in a disease-causing protein.

During biosynthesis, proteins can begin folding co-translationally to acquire their biologically-active structures. Folding, however, is an imperfect process and in many cases misfolding results in disease. Less is understood of how misfolding begins during biosynthesis. The human protein, alpha-1-antitrypsin (AAT) folds under kinetic control via a folding intermediate; its pathological variants readily form self-associated polymers at the site of synthesis, leading to alpha-1-antitrypsin deficiency. We observe that AAT nascent polypeptides stall during their biosynthesis, resulting in full-length nascent chains that remain bound to ribosome, forming a persistent ribosome-nascent chain complex (RNC) prior to release. We analyse the structure of these RNCs, which reveals compacted, partially-folded co-translational folding intermediates possessing molten-globule characteristics. We find that the highly-polymerogenic mutant, Z AAT, forms a distinct co-translational folding intermediate relative to wild-type. Its very modest structural differences suggests that the ribosome uniquely tempers the impact of deleterious mutations during nascent chain emergence. Following nascent chain release however, these co-translational folding intermediates guide post-translational folding outcomes thus suggesting that Z's misfolding is initiated from co-translational structure. Our findings demonstrate that co-translational folding intermediates drive how some proteins fold under kinetic control, and may thus also serve as tractable therapeutic targets for human disease.

Nascent chain dynamics and ribosome interactions within folded ribosome-nascent chain complexes observed by NMR spectroscopy.

The folding of many proteins can begin during biosynthesis on the ribosome and can be modulated by the ribosome itself. Such perturbations are generally believed to be mediated through interactions between the nascent chain and the ribosome surface, but despite recent progress in characterising interactions of unfolded states with the ribosome, and their impact on the initiation of co-translational folding, a complete quantitative analysis of interactions across both folded and unfolded states of a nascent chain has yet to be realised. Here we apply solution-state NMR spectroscopy to measure transverse proton relaxation rates for methyl groups in folded ribosome-nascent chain complexes of the FLN5 filamin domain. We observe substantial increases in relaxation rates for the nascent chain relative to the isolated domain, which can be related to changes in effective rotational correlation times using measurements of relaxation and cross-correlated relaxation in the isolated domain. Using this approach, we can identify interactions between the nascent chain and the ribosome surface, driven predominantly by electrostatics, and by measuring the change in these interactions as the subsequent FLN6 domain emerges, we may deduce their impact on the free energy landscapes associated with the co-translational folding process.

Interactions between nascent proteins and the ribosome surface inhibit co-translational folding.

Most proteins begin to fold during biosynthesis on the ribosome. It has been suggested that interactions between the emerging polypeptide and the ribosome surface might allow the ribosome itself to modulate co-translational folding. Here we combine protein engineering and NMR spectroscopy to characterize a series of interactions between the ribosome surface and unfolded nascent chains of the immunoglobulin-like FLN5 filamin domain. The strongest interactions are found for a C-terminal segment that is essential for folding, and we demonstrate quantitative agreement between the strength of this interaction and the energetics of the co-translational folding process itself. Mutations in this region that reduce the extent of binding result in a shift in the co-translational folding equilibrium towards the native state. Our results therefore demonstrate that a competition between folding and binding provides a simple, dynamic mechanism for the modulation of co-translational folding by the ribosome.

Full-length TDP-43 and its C-terminal domain form filaments in vitro having non-amyloid properties.

Accumulation of ubiquitin-positive, tau- and α-synuclein-negative intracellular inclusions of TDP-43 in the central nervous system represents the major hallmark correlated to amyotrophic lateral sclerosis (ALS) and frontotemporal lobar degeneration with ubiquitin-positive inclusions (FTLD-U). Such inclusions have variably been described as amorphous aggregates or more structured deposits having amyloid properties. Here we have purified full-length TDP-43 (FL TDP-43) and its C-terminal domain (Ct TDP-43) to investigate the morphological, structural and tinctorial features of aggregates formed in vitro by them at pH 7.4 and 37 °C. AFM images indicate that both protein variants show a tendency to form filaments. Moreover, we show that both FL TDP-43 and Ct TDP-43 filaments possess a largely disordered secondary structure, as ascertained by far-UV circular dichroism and Fourier transform infra-red spectroscopy, do not bind Congo red and induce a very weak increase of thioflavin T fluorescence, indicating the absence of a clear amyloid-like signature.

How Does the Ribosome Fold the Proteome?

Folding of polypeptides begins during their synthesis on ribosomes. This process has evolved as a means for the cell to maintain proteostasis, by mitigating the risk of protein misfolding and aggregation. The capacity to now depict this cellular feat at increasingly higher resolution is providing insight into the mechanistic determinants that promote successful folding. Emerging from these studies is the intimate interplay between protein translation and folding, and within this the ribosome particle is the key player. Its unique structural properties provide a specialized scaffold against which nascent polypeptides can begin to form structure in a highly coordinated, co-translational manner. Here, we examine how, as a macromolecular machine, the ribosome modulates the intrinsic dynamic properties of emerging nascent polypeptide chains and guides them toward their biologically active structures.

Probing the dynamic stalk region of the ribosome using solution NMR.

We describe an NMR approach based on the measurement of residual dipolar couplings (RDCs) to probe the structural and motional properties of the dynamic regions of the ribosome. Alignment of intact 70S ribosomes in filamentous bacteriophage enabled measurement of RDCs in the mobile C-terminal domain (CTD) of the stalk protein bL12. A structural refinement of this domain using the observed RDCs did not show large changes relative to the isolated protein in the absence of the ribosome, and we also found that alignment of the CTD was almost independent of the presence of the core ribosome particle, indicating that the inter-domain linker has significant flexibility. The nature of this linker was subsequently probed in more detail using a paramagnetic alignment strategy, which revealed partial propagation of alignment between neighbouring domains, providing direct experimental validation of a structural ensemble previously derived from SAXS and NMR relaxation measurements. Our results demonstrate the prospect of better characterising dynamical and functional regions of more challenging macromolecular machines and systems, for example ribosome-nascent chain complexes.

Systematic mapping of free energy landscapes of a growing filamin domain during biosynthesis.

Cotranslational folding (CTF) is a fundamental molecular process that ensures efficient protein biosynthesis and minimizes the formation of misfolded states. However, the complexity of this process makes it extremely challenging to obtain structural characterizations of CTF pathways. Here, we correlate observations of translationally arrested nascent chains with those of a systematic C-terminal truncation strategy. We create a detailed description of chain length-dependent free energy landscapes associated with folding of the FLN5 filamin domain, in isolation and on the ribosome, and thus, quantify a substantial destabilization of the native structure on the ribosome. We identify and characterize two folding intermediates formed in isolation, including a partially folded intermediate associated with the isomerization of a conserved cis proline residue. The slow folding associated with this process raises the prospect that neighboring unfolded domains might accumulate and misfold during biosynthesis. We develop a simple model to quantify the risk of misfolding in this situation and show that catalysis of folding by peptidyl-prolyl isomerases is sufficient to eliminate this hazard.

The ribosome and its role in protein folding: looking through a magnifying glass.

Protein folding, a process that underpins cellular activity, begins co-translationally on the ribosome. During translation, a newly synthesized polypeptide chain enters the ribosomal exit tunnel and actively interacts with the ribosome elements - the r-proteins and rRNA that line the tunnel - prior to emerging into the cellular milieu. While understanding of the structure and function of the ribosome has advanced significantly, little is known about the process of folding of the emerging nascent chain (NC). Advances in cryo-electron microscopy are enabling visualization of NCs within the exit tunnel, allowing early glimpses of the interplay between the NC and the ribosome. Once it has emerged from the exit tunnel into the cytosol, the NC (still attached to its parent ribosome) can acquire a range of conformations, which can be characterized by NMR spectroscopy. Using experimental restraints within molecular-dynamics simulations, the ensemble of NC structures can be described. In order to delineate the process of co-translational protein folding, a hybrid structural biology approach is foreseeable, potentially offering a complete atomic description of protein folding as it occurs on the ribosome.

A strategy for co-translational folding studies of ribosome-bound nascent chain complexes using NMR spectroscopy.

During biosynthesis on the ribosome, an elongating nascent polypeptide chain can begin to fold, in a process that is central to all living systems. Detailed structural studies of co-translational protein folding are now beginning to emerge; such studies were previously limited, at least in part, by the inherently dynamic nature of emerging nascent chains, which precluded most structural techniques. NMR spectroscopy is able to provide atomic-resolution information for ribosome-nascent chain complexes (RNCs), but it requires large quantities (≥10 mg) of homogeneous, isotopically labeled RNCs. Further challenges include limited sample working concentration and stability of the RNC sample (which contribute to weak NMR signals) and resonance broadening caused by attachment to the large (2.4-MDa) ribosomal complex. Here, we present a strategy to generate isotopically labeled RNCs in Escherichia coli that are suitable for NMR studies. Uniform translational arrest of the nascent chains is achieved using a stalling motif, and isotopically labeled RNCs are produced at high yield using high-cell-density E. coli growth conditions. Homogeneous RNCs are isolated by combining metal affinity chromatography (to isolate ribosome-bound species) with sucrose density centrifugation (to recover intact 70S monosomes). Sensitivity-optimized NMR spectroscopy is then applied to the RNCs, combined with a suite of parallel NMR and biochemical analyses to cross-validate their integrity, including RNC-optimized NMR diffusion measurements to report on ribosome attachment in situ. Comparative NMR studies of RNCs with the analogous isolated proteins permit a high-resolution description of the structure and dynamics of a nascent chain during its progressive biosynthesis on the ribosome.

Structural characterization of the interaction of α-synuclein nascent chains with the ribosomal surface and trigger factor.

The ribosome is increasingly becoming recognized as a key hub for integrating quality control processes associated with protein biosynthesis and cotranslational folding (CTF). The molecular mechanisms by which these processes take place, however, remain largely unknown, in particular in the case of intrinsically disordered proteins (IDPs). To address this question, we studied at a residue-specific level the structure and dynamics of ribosome-nascent chain complexes (RNCs) of α-synuclein (αSyn), an IDP associated with Parkinson's disease (PD). Using solution-state nuclear magnetic resonance (NMR) spectroscopy and coarse-grained molecular dynamics (MD) simulations, we find that, although the nascent chain (NC) has a highly disordered conformation, its N-terminal region shows resonance broadening consistent with interactions involving specific regions of the ribosome surface. We also investigated the effects of the ribosome-associated molecular chaperone trigger factor (TF) on αSyn structure and dynamics using resonance broadening to define a footprint of the TF-RNC interactions. We have used these data to construct structural models that suggest specific ways by which emerging NCs can interact with the biosynthesis and quality control machinery.

Two-Dimensional NMR Lineshape Analysis.

NMR titration experiments are a rich source of structural, mechanistic, thermodynamic and kinetic information on biomolecular interactions, which can be extracted through the quantitative analysis of resonance lineshapes. However, applications of such analyses are frequently limited by peak overlap inherent to complex biomolecular systems. Moreover, systematic errors may arise due to the analysis of two-dimensional data using theoretical frameworks developed for one-dimensional experiments. Here we introduce a more accurate and convenient method for the analysis of such data, based on the direct quantum mechanical simulation and fitting of entire two-dimensional experiments, which we implement in a new software tool, TITAN (TITration ANalysis). We expect the approach, which we demonstrate for a variety of protein-protein and protein-ligand interactions, to be particularly useful in providing information on multi-step or multi-component interactions.

A structural ensemble of a ribosome-nascent chain complex during cotranslational protein folding.

Although detailed pictures of ribosome structures are emerging, little is known about the structural and cotranslational folding properties of nascent polypeptide chains at the atomic level. Here we used solution-state NMR spectroscopy to define a structural ensemble of a ribosome-nascent chain complex (RNC) formed during protein biosynthesis in Escherichia coli, in which a pair of immunoglobulin-like domains adopts a folded N-terminal domain (FLN5) and a disordered but compact C-terminal domain (FLN6). To study how FLN5 acquires its native structure cotranslationally, we progressively shortened the RNC constructs. We found that the ribosome modulates the folding process, because the complete sequence of FLN5 emerged well beyond the tunnel before acquiring native structure, whereas FLN5 in isolation folded spontaneously, even when truncated. This finding suggests that regulating structure acquisition during biosynthesis can reduce the probability of misfolding, particularly of homologous domains.