Direct observation of prion-like propagation of protein misfolding templated by pathogenic mutants.

Many neurodegenerative diseases feature misfolded proteins that propagate via templated conversion of natively folded molecules. However, crucial questions about how such prion-like conversion occurs and what drives it remain unsolved, partly because technical challenges have prevented direct observation of conversion for any protein. We observed prion-like conversion in single molecules of superoxide dismutase-1 (SOD1), whose misfolding is linked to amyotrophic lateral sclerosis. Tethering pathogenic misfolded SOD1 mutants to wild-type molecules held in optical tweezers, we found that the mutants vastly increased misfolding of the wild-type molecule, inducing multiple misfolded isoforms. Crucially, the pattern of misfolding was the same in the mutant and converted wild-type domains and varied when the misfolded mutant was changed, reflecting the templating effect expected for prion-like conversion. Ensemble measurements showed decreased enzymatic activity in tethered heterodimers as conversion progressed, mirroring the single-molecule results. Antibodies sensitive to disease-specific epitopes bound to the converted protein, implying that conversion produced disease-relevant misfolded conformers.

Mast Cell Proteases Cleave Prion Proteins and a Recombinant Ig against PrP Can Activate Human Mast Cells.

IgE Abs, best known for their role in allergic reactions, have only rarely been used in immunotherapies. Nevertheless, they offer a potential alternative to the more commonly used IgGs. The affinity of IgE Ag binding influences the type of response from mast cells, so any immunotherapies using IgEs must balance Ag affinity with desired therapeutic effect. One potential way to harness differential binding affinities of IgE is in protein aggregation diseases, where low-affinity binding of endogenous proteins is preferred, but enhanced binding of clusters of disease-associated aggregated proteins could target responses to the sites of disease. For this reason, we sought to create a low-affinity IgE against the prion protein (PrP), which exists in an endogenous monomeric state but can misfold into aggregated states during the development of prion disease. First, we determined that mast cell proteases tryptase and cathepsin G were capable of degrading PrP. Then we engineered a recombinant IgE Ab directed against PrP from the V region of a PrP-specific IgG and tested its activation of the human mast cell line LAD2. The αPrP IgE bound LAD2 through Fc receptors. Crosslinking receptor-bound αPrP IgE activated SYK and ERK phosphorylation, caused Fc receptor internalization, and resulted in degranulation. This work shows that a recombinant αPrP IgE can activate LAD2 cells to release enzymes that can degrade PrP, suggesting that IgE may be useful in targeting diseases that involve protein aggregation.

Atomistic structure of the SARS-CoV-2 pseudoknot in solution from SAXS-driven molecular dynamics.

SARS-CoV-2 depends on -1 programmed ribosomal frameshifting (-1 PRF) to express proteins essential for its replication. The RNA pseudoknot stimulating -1 PRF is thus an attractive drug target. However, the structural models of this pseudoknot obtained from cryo-EM and crystallography differ in some important features, leaving the pseudoknot structure unclear. We measured the solution structure of the pseudoknot using small-angle X-ray scattering (SAXS). The measured profile did not agree with profiles computed from the previously solved structures. Beginning with each of these solved structures, we used the SAXS data to direct all atom molecular dynamics (MD) simulations to improve the agreement in profiles. In all cases, this refinement resulted in a bent conformation that more closely resembled the cryo-EM structures than the crystal structure. Applying the same approach to a point mutant abolishing -1 PRF revealed a notably more bent structure with reoriented helices. This work clarifies the dynamic structures of the SARS-CoV-2 pseudoknot in solution.

Modelling the structures of frameshift-stimulatory pseudoknots from representative bat coronaviruses.

Coronaviruses (CoVs) use -1 programmed ribosomal frameshifting stimulated by RNA pseudoknots in the viral genome to control expression of enzymes essential for replication, making CoV pseudoknots a promising target for anti-coronaviral drugs. Bats represent one of the largest reservoirs of CoVs and are the ultimate source of most CoVs infecting humans, including those causing SARS, MERS, and COVID-19. However, the structures of bat-CoV frameshift-stimulatory pseudoknots remain largely unexplored. Here we use a combination of blind structure prediction followed by all-atom molecular dynamics simulations to model the structures of eight pseudoknots that, together with the SARS-CoV-2 pseudoknot, are representative of the range of pseudoknot sequences in bat CoVs. We find that they all share some key qualitative features with the pseudoknot from SARS-CoV-2, notably the presence of conformers with two distinct fold topologies differing in whether or not the 5' end of the RNA is threaded through a junction, and similar conformations for stem 1. However, they differed in the number of helices present, with half sharing the 3-helix architecture of the SARS-CoV-2 pseudoknot but two containing 4 helices and two others only 2. These structure models should be helpful for future work studying bat-CoV pseudoknots as potential therapeutic targets.

Observing the base-by-base search for native structure along transition paths during the folding of single nucleic acid hairpins.

Biomolecular folding involves searching among myriad possibilities for the native conformation, but the elementary steps expected from theory for this search have never been detected directly. We probed the dynamics of folding at high resolution using optical tweezers, measuring individual trajectories as nucleic acid hairpins passed through the high-energy transition states that dominate kinetics and define folding mechanisms. We observed brief but ubiquitous pauses in the transition states, with a dwell time distribution that matched microscopic theories of folding quantitatively. The sequence dependence suggested that pauses were dominated by microbarriers from nonnative conformations during the search by each nucleotide residue for the native base-pairing conformation. Furthermore, the pauses were position dependent, revealing subtle local variations in energy-landscape roughness and allowing the diffusion coefficient describing the microscopic dynamics within the barrier to be found without reconstructing the shape of the energy landscape. These results show how high-resolution measurements can elucidate key microscopic events during folding to test fundamental theories of folding.

Unfolded and intermediate states of PrP play a key role in the mechanism of action of an antiprion chaperone.

Prion and prion-like diseases involve the propagation of misfolded protein conformers. Small-molecule pharmacological chaperones can inhibit propagated misfolding, but how they interact with disease-related proteins to prevent misfolding is often unclear. We investigated how pentosan polysulfate (PPS), a polyanion with antiprion activity in vitro and in vivo, interacts with mammalian prion protein (PrP) to alter its folding. Calorimetry showed that PPS binds two sites on natively folded PrP, but one PPS molecule can bind multiple PrP molecules. Force spectroscopy measurements of single PrP molecules showed PPS stabilizes not only the native fold of PrP but also many different partially folded intermediates that are not observed in the absence of PPS. PPS also bound tightly to unfolded segments of PrP, delaying refolding. These observations imply that PPS can act through multiple possible modes, inhibiting misfolding not only by stabilizing the native fold or sequestering natively folded PrP into aggregates, as proposed previously, but also by binding to partially or fully unfolded states that play key roles in mediating misfolding. These results underline the likely importance of unfolded states as critical intermediates on the prion conversion pathway.

Nonlinear effects in optical trapping of titanium dioxide and diamond nanoparticles.

Optical trapping in biophysics typically uses micron-scale beads made of materials like polystyrene or glass to probe the target of interest. Using smaller beads made of higher-index materials could increase the time resolution of these measurements. We characterized the trapping of nanoscale beads made of diamond and titanium dioxide (TiO2) in a single-beam gradient trap. Calculating theoretical expectations for the trapping stiffness of these beads, we found good agreement with measured values. Trap stiffness was significantly higher for TiO2 beads, owing to notable enhancement from nonlinear optical effects, not previously observed for continuous-wave trapping. Trap stiffness was over 6-fold higher for TiO2 beads than polystyrene beads of similar size at 70 mW laser power. These results suggest that diamond and TiO2 nanobeads can be used to improve time resolution in optical tweezers measurements.

Mechanical strength of RNA knot in Zika virus protects against cellular defenses.

Unusual knot-like structures recently discovered in viral exoribonuclease-resistant RNAs (xrRNAs) prevent digestion by host RNases to create subgenomic RNAs enhancing infection and pathogenicity. xrRNAs are proposed to prevent digestion through mechanical resistance to unfolding. However, their unfolding force has not been measured, and the factors determining RNase resistance are unclear. Furthermore, how these knots fold remains unknown. Unfolding a Zika virus xrRNA with optical tweezers revealed that it was the most mechanically stable RNA yet observed. The knot formed by threading the 5' end into a three-helix junction before pseudoknot interactions closed a ring around it. The pseudoknot and tertiary contacts stabilizing the threaded 5' end were both required to generate extreme force resistance, whereas removing a 5'-end contact produced a low-force knot lacking RNase resistance. These results indicate mechanical resistance plays a central functional role, with the fraction of molecules forming extremely high-force knots determining the RNase resistance level.

Modeling the structure of the frameshift-stimulatory pseudoknot in SARS-CoV-2 reveals multiple possible conformers.

The coronavirus causing the COVID-19 pandemic, SARS-CoV-2, uses -1 programmed ribosomal frameshifting (-1 PRF) to control the relative expression of viral proteins. As modulating -1 PRF can inhibit viral replication, the RNA pseudoknot stimulating -1 PRF may be a fruitful target for therapeutics treating COVID-19. We modeled the unusual 3-stem structure of the stimulatory pseudoknot of SARS-CoV-2 computationally, using multiple blind structural prediction tools followed by µs-long molecular dynamics simulations. The results were compared for consistency with nuclease-protection assays and single-molecule force spectroscopy measurements of the SARS-CoV-1 pseudoknot, to determine the most likely conformations. We found several possible conformations for the SARS-CoV-2 pseudoknot, all having an extended stem 3 but with different packing of stems 1 and 2. Several conformations featured rarely-seen threading of a single strand through junctions formed between two helices. These structural models may help interpret future experiments and support efforts to discover ligands inhibiting -1 PRF in SARS-CoV-2.

Measuring the average shape of transition paths during the folding of a single biological molecule.

Transition paths represent the parts of a reaction where the energy barrier separating products and reactants is crossed. They are essential to understanding reaction mechanisms, yet many of their properties remain unstudied. Here, we report measurements of the average shape of transition paths, studying the folding of DNA hairpins as a model system for folding reactions. Individual transition paths were detected in the folding trajectories of hairpins with different sequences held under tension in optical tweezers, and path shapes were computed by averaging all transitions in the time domain, 〈t(x)〉, or by averaging transitions of a given duration in the extension domain, 〈x(t|τ)〉 τ Whereas 〈t(x)〉 was close to straight, with only a subtle curvature, 〈x(t|τ)〉 τ had more pronounced curvature that fit well to theoretical expectations for the dominant transition path, returning diffusion coefficients similar to values obtained previously from independent methods. Simulations suggested that 〈t(x)〉 provided a less reliable representation of the path shape than 〈x(t|τ)〉 τ , because it was far more sensitive to the effects of coupling the molecule to the experimental force probe. Intriguingly, the path shape variance was larger for some hairpins than others, indicating sequence-dependent changes in the diversity of transition paths reflective of differences in the character of the energy barriers, such as the width of the barrier saddle-point or the presence of parallel paths through multiple barriers between the folded and unfolded states. These studies of average path shapes point the way forward for probing the rich information contained in path shape fluctuations.

Complex dynamics under tension in a high-efficiency frameshift stimulatory structure.

Specific structures in mRNA can stimulate programmed ribosomal frameshifting (PRF). PRF efficiency can vary enormously between different stimulatory structures, but the features that lead to efficient PRF stimulation remain uncertain. To address this question, we studied the structural dynamics of the frameshift signal from West Nile virus (WNV), which stimulates -1 PRF at very high levels and has been proposed to form several different structures, including mutually incompatible pseudoknots and a double hairpin. Using optical tweezers to apply tension to single mRNA molecules, mimicking the tension applied by the ribosome during PRF, we found that the WNV frameshift signal formed an unusually large number of different metastable structures, including all of those previously proposed. From force-extension curve measurements, we mapped 2 mutually exclusive pathways for the folding, each encompassing multiple intermediates. We identified the intermediates in each pathway from length changes and the effects of antisense oligomers blocking formation of specific contacts. Intriguingly, the number of transitions between the different conformers of the WNV frameshift signal was maximal in the range of forces applied by the ribosome during -1 PRF. Furthermore, the occupancy of the pseudoknotted conformations was far too low for static pseudoknots to account for the high levels of -1 PRF. These results support the hypothesis that conformational heterogeneity plays a key role in frameshifting and suggest that transitions between different conformers under tension are linked to efficient PRF stimulation.

Internalization of α-synuclein oligomers into SH-SY5Y cells.

Aggregates of misfolded α-synuclein are a distinctive feature of Parkinson's disease. Small oligomers of α-synuclein are thought to be an important neurotoxic agent, and α-synuclein aggregates exhibit prion-like behavior, propagating misfolding between cells. α-Synuclein is internalized by both passive diffusion and active uptake mechanisms, but how uptake varies with the size of the oligomer is less clear. We explored how α-synuclein internalization into live SH-SY5Y cells varied with oligomer size by comparing the uptake of fluorescently labeled monomers to that of engineered tandem dimers and tetramers. We found that these α-synuclein constructs were internalized primarily through endocytosis. Oligomer size had little effect on their internalization pathway, whether they were added individually or together. Measurements of co-localization of the α-synuclein constructs with fluorescent markers for early endosomes and lysosomes showed that most of the α-synuclein entered endocytic compartments, in which they were probably degraded. Treatment of the cells with the Pitstop inhibitor suggested that most of the oligomers were internalized by the clathrin-mediated pathway.

Structural and functional conservation of the programmed -1 ribosomal frameshift signal of SARS coronavirus 2 (SARS-CoV-2).

Approximately 17 years after the severe acute respiratory syndrome coronavirus (SARS-CoV) epidemic, the world is currently facing the COVID-19 pandemic caused by SARS corona virus 2 (SARS-CoV-2). According to the most optimistic projections, it will take more than a year to develop a vaccine, so the best short-term strategy may lie in identifying virus-specific targets for small molecule-based interventions. All coronaviruses utilize a molecular mechanism called programmed -1 ribosomal frameshift (-1 PRF) to control the relative expression of their proteins. Previous analyses of SARS-CoV have revealed that it employs a structurally unique three-stemmed mRNA pseudoknot that stimulates high -1 PRF rates and that it also harbors a -1 PRF attenuation element. Altering -1 PRF activity impairs virus replication, suggesting that this activity may be therapeutically targeted. Here, we comparatively analyzed the SARS-CoV and SARS-CoV-2 frameshift signals. Structural and functional analyses revealed that both elements promote similar -1 PRF rates and that silent coding mutations in the slippery sites and in all three stems of the pseudoknot strongly ablate -1 PRF activity. We noted that the upstream attenuator hairpin activity is also functionally retained in both viruses, despite differences in the primary sequence in this region. Small-angle X-ray scattering analyses indicated that the pseudoknots in SARS-CoV and SARS-CoV-2 have the same conformation. Finally, a small molecule previously shown to bind the SARS-CoV pseudoknot and inhibit -1 PRF was similarly effective against -1 PRF in SARS-CoV-2, suggesting that such frameshift inhibitors may be promising lead compounds to combat the current COVID-19 pandemic.

Conformational Shannon Entropy of mRNA Structures from Force Spectroscopy Measurements Predicts the Efficiency of -1 Programmed Ribosomal Frameshift Stimulation.

-1 programmed ribosomal frameshifting (-1 PRF) is stimulated by structures in messenger RNA (mRNA), but the factors determining -1 PRF efficiency are unclear. We show that -1 PRF efficiency varies directly with the conformational heterogeneity of the stimulatory structure, quantified as the Shannon entropy of the state occupancy, for a panel of stimulatory structures with efficiencies from 2% to 80%. The correlation is force dependent and vanishes at forces above those applied by the ribosome. These results support the hypothesis that heterogeneous conformational dynamics are a key factor in stimulating -1 PRF.

Direct measurement of sequence-dependent transition path times and conformational diffusion in DNA duplex formation.

The conformational diffusion coefficient, D, sets the timescale for microscopic structural changes during folding transitions in biomolecules like nucleic acids and proteins. D encodes significant information about the folding dynamics such as the roughness of the energy landscape governing the folding and the level of internal friction in the molecule, but it is challenging to measure. The most sensitive measure of D is the time required to cross the energy barrier that dominates folding kinetics, known as the transition path time. To investigate the sequence dependence of D in DNA duplex formation, we measured individual transition paths from equilibrium folding trajectories of single DNA hairpins held under tension in high-resolution optical tweezers. Studying hairpins with the same helix length but with G:C base-pair content varying from 0 to 100%, we determined both the average time to cross the transition paths, τtp, and the distribution of individual transit times, PTP(t). We then estimated D from both τtp and PTP(t) from theories assuming one-dimensional diffusive motion over a harmonic barrier. τtp decreased roughly linearly with the G:C content of the hairpin helix, being 50% longer for hairpins with only A:T base pairs than for those with only G:C base pairs. Conversely, D increased linearly with helix G:C content, roughly doubling as the G:C content increased from 0 to 100%. These results reveal that G:C base pairs form faster than A:T base pairs because of faster conformational diffusion, possibly reflecting lower torsional barriers, and demonstrate the power of transition path measurements for elucidating the microscopic determinants of folding.

Probing the Basis of α-Synuclein Aggregation by Comparing Simulations to Single-Molecule Experiments.

Intrinsically disordered proteins often play an important role in protein aggregation. However, it is challenging to determine the structures and interactions that drive the early stages of aggregation because they are transient and obscured in a heterogeneous mixture of disordered states. Even computational methods are limited because the lack of ordered structure makes it difficult to ensure that the relevant conformations are sampled. We address these challenges by integrating atomistic simulations with high-resolution single-molecule measurements reported previously, using the measurements to help discern which parts of the disordered ensemble of structures in the simulations are most probable while using the simulations to identify residues and interactions that are important for oligomer stability. This approach was applied to α-synuclein, an intrinsically disordered protein that aggregates in the context of Parkinson's disease. We simulated single-molecule pulling experiments on dimers, the minimal oligomer, and compared them to force spectroscopy measurements. Force-extension curves were simulated starting from a set of 66 structures with substantial structured content selected from the ensemble of dimer structures generated at zero force via Monte Carlo simulations. The pattern of contour length changes as the structures unfolded through intermediate states was compared to the results from optical trapping measurements on the same dimer to discern likely structures occurring in the measurements. Simulated pulling curves were generally consistent with experimental data but with a larger number of transient intermediates. We identified an ensemble of ß-rich dimer structures consistent with the experimental data from which dimer interfaces could be deduced. These results suggest specific druggable targets in the structural motifs of α-synuclein that may help prevent the earliest steps of oligomerization.

Conformational dynamics of the frameshift stimulatory structure in HIV-1.

Programmed ribosomal frameshifting (PRF) in HIV-1 is thought to be stimulated by a hairpin in the mRNA, although a pseudoknot-like triplex has also been proposed. Because the conformational dynamics of the stimulatory structure under tension applied by the ribosomal helicase during translation may play an important role in PRF, we used optical tweezers to apply tension to the HIV stimulatory structure and monitor its unfolding and refolding dynamics. The folding and unfolding kinetics and energy landscape of the hairpin were measured by ramping the force on the hairpin up and down, providing a detailed biophysical characterization. Unexpectedly, whereas unfolding reflected the simple two-state behavior typical of many hairpins, refolding was more complex, displaying significant heterogeneity. Evidence was found for multiple refolding pathways as well as previously unsuspected, partially folded intermediates. Measuring a variant mRNA containing only the sequence required to form the proposed triplex, it behaved largely in the same way. Nonetheless, very rarely, high-force unfolding events characteristic of pseudoknot-like structures were observed. The rare occurrence of the triplex suggests that the hairpin is the functional stimulatory structure. The unusual heterogeneity of the hairpin dynamics under tension suggests a possible functional role in PRF similar to the dynamics of other stimulatory structures.

Memory effects in single-molecule force spectroscopy measurements of biomolecular folding.

Folding is generally assumed to be a Markov process, without memory. When the molecular motion is coupled to that of a probe as in single-molecule force spectroscopy (SMFS) experiments, however, theory predicts that the coupling to a second Markov process should induce memory when monitoring a projection of the full multi-dimensional motion onto a reduced coordinate. We developed a method to evaluate the time constant of the induced memory from its effects on the autocorrelation function, which can be readily determined from experimental data. Applying this method to both simulated SMFS measurements and experimental trajectories of DNA hairpin folding measured by optical tweezers as a model system, we validated the prediction that the linker induces memory. For these measurements, the timescale of the induced memory was found to be similar to the time required for the force probe to respond to changes in the molecule, and in the regime where the experimentally observed dynamics were not significantly perturbed by probe-molecule coupling artifacts. Memory effects are thus a general feature of SMFS measurements induced by the mechanical connection between the molecule and force probe that should be considered when interpreting experimental data.

Molecular free energy profiles from force spectroscopy experiments by inversion of observed committors.

In single-molecule force spectroscopy experiments, a biomolecule is attached to a force probe via polymer linkers and the total extension of the molecule plus apparatus is monitored as a function of time. In a typical unfolding experiment at constant force, the total extension jumps between two values that correspond to the folded and unfolded states of the molecule. For several biomolecular systems, the committor, which is the probability to fold starting from a given extension, has been used to extract the molecular activation barrier (a technique known as "committor inversion"). In this work, we study the influence of the force probe, which is much larger than the molecule being measured, on the activation barrier obtained by committor inversion. We use a two-dimensional framework in which the diffusion coefficient of the molecule and of the pulling device can differ. We systematically study the free energy profile along the total extension obtained from the committor by numerically solving the Onsager equation and using Brownian dynamics simulations. We analyze the dependence of the extracted barrier on the linker stiffness, molecular barrier height, and diffusion anisotropy and, thus, establish the range of validity of committor inversion. Along the way, we showcase the committor of 2-dimensional diffusive models and illustrate how it is affected by barrier asymmetry and diffusion anisotropy.

High-Precision Single-Molecule Characterization of the Folding of an HIV RNA Hairpin by Atomic Force Microscopy.

The folding of RNA into a wide range of structures is essential for its diverse biological functions from enzymatic catalysis to ligand binding and gene regulation. The unfolding and refolding of individual RNA molecules can be probed by single-molecule force spectroscopy (SMFS), enabling detailed characterization of the conformational dynamics of the molecule as well as the free-energy landscape underlying folding. Historically, high-precision SMFS studies of RNA have been limited to custom-built optical traps. Although commercial atomic force microscopes (AFMs) are widely deployed and offer significant advantages in ease-of-use over custom-built optical traps, traditional AFM-based SMFS lacks the sensitivity and stability to characterize individual RNA molecules precisely. Here, we developed a high-precision SMFS assay to study RNA folding using a commercial AFM and applied it to characterize a small RNA hairpin from HIV that plays a key role in stimulating programmed ribosomal frameshifting. We achieved rapid data acquisition in a dynamic assay, unfolding and then refolding the same individual hairpin more than 1,100 times in 15 min. In comparison to measurements using optical traps, our AFM-based assay featured a stiffer force probe and a less compliant construct, providing a complementary measurement regime that dramatically accelerated equilibrium folding dynamics. Not only did kinetic analysis of equilibrium trajectories of the HIV RNA hairpin yield the traditional parameters used to characterize folding by SMFS (zero-force rate constants and distances to the transition state), but we also reconstructed the full 1D projection of the folding free-energy landscape comparable to state-of-the-art studies using dual-beam optical traps, a first for this RNA hairpin and AFM studies of nucleic acids in general. Looking forward, we anticipate that the ease-of-use of our high-precision assay implemented on a commercial AFM will accelerate studying folding of diverse nucleic acid structures.