Prions are unusual protein assemblies that propagate their conformationally-encoded information in absence of nucleic acids. The first prion identified, the scrapie isoform (PrP Sc ) of the cellular prion protein (PrP C ), caused epidemic and epizootic episodes [ 1 ]. Most aggregates of other misfolding-prone proteins are amyloids, often arranged in a Parallel-In-Register-β-Sheet (PIRIBS) [ 2 ] or β-solenoid conformations [ 3 ]. Similar folding models have also been proposed for PrP Sc , although none of these have been confirmed experimentally. Recent cryo-electron microscopy (cryo-EM) and X-ray fiber-diffraction studies provided evidence that PrP Sc is structured as a 4-rung β-solenoid (4RβS) [ 4 , 5 ]. Here, we combined different experimental data and computational techniques to build the first physically-plausible, atomic resolution model of mouse PrP Sc , based on the 4RβS architecture. The stability of this new PrP Sc model, as assessed by Molecular Dynamics (MD) simulations, was found to be comparable to that of the prion forming domain of Het-s, a naturally-occurring β-solenoid. Importantly, the 4RβS arrangement allowed the first simulation of the sequence of events underlying PrP C conversion into PrP Sc . This study provides the most updated, experimentally-driven and physically-coherent model of PrP Sc , together with an unprecedented reconstruction of the mechanism underlying the self-catalytic propagation of prions.

The ever increasing computer power, together with the improved accuracy of atomistic force fields, enables researchers to investigate biological systems at the molecular level with remarkable detail. However, the relevant length and time scales of many processes of interest are still hardly within reach even for state-of-the-art hardware, thus leaving important questions often unanswered. The computer-aided investigation of many biological physics problems thus largely benefits from the usage of coarse-grained models, that is, simplified representations of a molecule at a level of resolution that is lower than atomistic. A plethora of coarse-grained models have been developed, which differ most notably in their granularity; this latter aspect determines one of the crucial open issues in the field, i.e. the identification of an optimal degree of coarsening, which enables the greatest simplification at the expenses of the smallest information loss. In this review, we present the problem of coarse-grained modeling in biophysics from the viewpoint of system representation and information content. In particular, we discuss two distinct yet complementary aspects of protein modeling: on the one hand, the relationship between the resolution of a model and its capacity of accurately reproducing the properties of interest; on the other hand, the possibility of employing a lower resolution description of a detailed model to extract simple, useful, and intelligible information from the latter.

Recent computational advancements in the simulation of biochemical processes allow investigating the mechanisms involved in protein regulation with realistic physics-based models, at an atomistic level of resolution. These techniques allowed us to design a drug discovery approach, named Pharmacological Protein Inactivation by Folding Intermediate Targeting (PPI-FIT), based on the rationale of negatively regulating protein levels by targeting folding intermediates. Here, PPI-FIT was tested for the first time on the cellular prion protein (PrP), a cell surface glycoprotein playing a key role in fatal and transmissible neurodegenerative pathologies known as prion diseases. We predicted the all-atom structure of an intermediate appearing along the folding pathway of PrP and identified four different small molecule ligands for this conformer, all capable of selectively lowering the load of the protein by promoting its degradation. Our data support the notion that the level of target proteins could be modulated by acting on their folding pathways, implying a previously unappreciated role for folding intermediates in the biological regulation of protein expression.

Prions are unusual protein assemblies that propagate their conformationally-encoded information in absence of nucleic acids. The first prion identified, the scrapie isoform (PrP Sc ) of the cellular prion protein (PrP C ), is the only one known to cause epidemic and epizootic episodes(1). Most aggregates of other misfolding-prone proteins are amyloids, often arranged in a Parallel-In-Register-β-Sheet (PIRIBS)(2) or βsolenoid conformations(3). Similar folding models have also been proposed for PrP Sc , although none of these have been confirmed experimentally. Recent cryo-electron microscopy (cryo-EM) and X-ray fiberdiffraction studies provided evidence that PrP Sc is structured as a 4-rung β-solenoid (4RβS)(4, 5). Here, we combined different experimental data and computational techniques to build the first physically-plausible, atomic resolution model of mouse PrP Sc , based on the 4RβS architecture. The stability of this new PrP Sc model, as assessed by Molecular Dynamics (MD) simulations, was found to be comparable to that of the prion forming domain of Het-s, a naturally-occurring β-solenoid. Importantly, the 4RβS arrangement allowed the first simulation of the sequence of events underlying PrP C conversion into PrP Sc . Our results provide the most updated, experimentally-driven and physically-coherent model of PrP Sc , together with an unprecedented reconstruction of the mechanism underlying the self-catalytic propagation of prions. SignificanceSince the original hypothesis by Stanley Prusiner, prions have represented enigmatic agents diverging from the classical concept of genetic inheritance. However, the structure of PrP Sc , the infectious isoform of the cellular prion protein (PrP C ), has so far remained elusive, mostly due to technical challenges posed by its aggregation propensity. Here, we present a new high resolution model of PrP Sc derived from the integration of a wide array of recent experimental constraints. By coupling the information of such model with a newly developed computational method, we reconstructed for the first time the conformational transition of PrP C to PrP Sc . This study offers a unique workbench for designing therapeutics against prion diseases, and a physically-plausible mechanism explaining how protein conformation could self-propagate.

The affinity of an antibody for its antigen is primarily determined by the specific sequence and structural arrangement of the complementarity-determining regions (CDRs). Recent evidence, however, points toward a nontrivial relation between the CDR and distal sites: variations in the binding strengths have been observed upon mutating residues separated from the paratope by several nanometers, thus suggesting the existence of a communication network within antibodies, whose extension and relevance might be deeper than insofar expected. In this work, we test this hypothesis by means of molecular dynamics (MD) simulations of the IgG4 monoclonal antibody pembrolizumab, an approved drug that targets the programmed cell death protein 1 (PD-1). The molecule is simulated in both the apo and holo states, totalling 4 μs of MD trajectory. The analysis of these simulations shows that the bound antibody explores a restricted range of conformations with respect to the apo one, and that the global conformation of the molecule correlates with that of the CDR. These results support the hypothesis that pembrolizumab featues a multi-scale hierarchy of intertwined global and local conformational changes. The analysis pipeline developed in this work is general, and it can help shed further light on the mechanistic aspects of antibody function.

Deformed templating is the process by which self-replicating protein conformations with a given cross-β folding pattern can seed formation of an alternative self-replicating state with different cross-β folding pattern. In particular, uninfectious but propagative PrP amyloid can transform into a bona fide infectious conformer, PrP Sc through deformed templating. The process can take many rounds of replication (if taking place in vitro ) or even several passages of the evolving PrP conformers through successive brains if in vivo , through experimental transmission. In all cases, deformed templating involves a forced conversion in which there is a mismatch between the template and the substrate and/or the templating environment, typically a recombinant PrP amyloid, adept at converting recombinant PrP under denaturing conditions (e.g., presence of chaotropic agents), encountering a glycosylated, GPI-anchored PrP C substrate under physiological conversion conditions. Deformed templating is characterized by emergence of intermediate conformers that exhibit biochemical characteristics that are intermediate between those of the initial PrP amyloid and the final PrP Sc conformers. Here, we took advantage of the recent elucidation of the structure of a PrP amyloid by cryo-EM and the availability of a physically plausible atomistic model of PrP Sc that we have recently proposed. Using modeling and Molecular Dynamics (MD) approaches, we built a complete molecular modelization of deformed templating, including an atomistic model of a glycosylated intermediate conformer and a modified model of PrP Sc . Among other unanticipated outcomes, our results show that fully glycosylated PrP can be stacked in-register, and how 4-rung β-solenoid (4RβS) PrP architectures can share key structural motifs with parallel-in register intermolecular sheet (PIRIBS) PrP amyloids. Our results shed light on the mechanisms of prion replication.

Recent computational advancements in the simulation of biochemical processes allow investigating the mechanisms involved in protein regulation with realistic physics-based models, at an atomistic level of resolution. Using these techniques to study the negative regulation of the androgen receptor (AR), we discovered a key functional role played by non-native metastable states appearing along the folding pathway of this protein. This unexpected observation inspired us to design a completely novel drug discovery approach, named Pharmacological Protein Inactivation by Folding Intermediate Targeting (PPI-FIT), based on the rationale of negatively regulating protein expression by targeting folding intermediates. Here, PPI-FIT was tested for the first time on the cellular prion protein (PrP), a cell surface glycoprotein playing a key role in fatal and transmissible neurodegenerative pathologies known as prion diseases. We predicted the all-atom structure of an intermediate appearing along the folding pathway of PrP, and identified four different small molecule ligands for this conformer, all capable of selectively lowering the expression of the protein by promoting its degradation. Our data support the notion that the level of target proteins could be modulated by acting on their folding pathways, implying a previously unappreciated role for folding intermediates in the biological regulation of protein expression. INTRODUCTIONProtein expression and function in eukaryotic cells are tightly harmonized processes modulated by the combination of different layers of regulation. These may occur at the level of gene transcription, processing, stability, and translation of the mRNA as well as by assembly, post-translational modifications, sorting, recycling, and degradation of the corresponding polypeptide 1 . In addition, the expression of a small subset of proteins is known to be regulated by the presence of specific ligands, which are required along the folding pathway to reach the native, functional state 2-7 . For example, the folding of the kinase-inducible domain of the cAMP-response-element-binding protein is known to proceed only upon interaction with the CREB-binding protein 8 . Other typical examples of liganddependent folding mechanism include nuclear receptors, such as estrogen and androgen receptors 9 . The integration between these pathways and the protein quality control machinery, deputed to avoid the production and accumulation of aberrantly folded proteins, is known as proteostasis 1,10 . Mechanisms by which proteostasis is ensured include chaperone-assisted protein folding as well as re-routing and degradation of misfolded protein conformers. These processes play a fundamental role during development, aging, tolerance to environmental stresses, and minimize alterations of proteome homeostasis induced by pathogens 11 . Consistently, a large percentage of human pathologies are linked to alterations of proteostasis, including a broad spectrum of age-related brain disorders, known as neurodegenerative diseases, ...

No abstract