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. Prions are unusual infectious pathogens that do not contain any nucleic acid. They consist of assemblies of misfolded proteins. The scrapie isoform of the mammalian prion protein, PrP^Sc, is the most notorious prion, and is responsible for deadly neurodegenerative diseases affecting humans, like Creutzfeldt-Jakob disease, and animals, such as bovine spongiform encephalopathy (“mad cow disease”) and chronic wasting disease affecting elk and deer in North America and, more recently, Europe). Understanding the structure (“shape”) of the PrP^Sc prion is critical to understand how it propagates. We have created a very detailed model of PrP^Sc, which includes all its atoms, using computational techniques. The model resembles a 4-rung cork-screw. The model is stable and agrees with all available experimental data on PrP^Sc. This structure allowed us to model for the first time the process of prion propagation at high resolution. These data inspire several new hypotheses to elucidate prion biology and design therapeutics for prion diseases

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.

The formation of a tetrameric assembly is essential for the ability of the tumor suppressor protein p53 to act as a transcription factor. Such a quaternary conformation is driven by a specific tetramerization domain, separated from the central DNA-binding domain by a flexible linker. Despite the distance, functional crosstalk between the two domains has been reported. This phenomenon can explain the pathogenicity of some inherited or somatically acquired mutations in the tetramerization domain, including the widespread R337H missense mutation present in the population in south Brazil. In this work, we combined computational predictions through extended all-atom molecular dynamics simulations with functional assays in a genetically defined yeast-based model system to reveal structural features of p53 tetramerization domains and their transactivation capacity and specificity. In addition to the germline and cancer-associated R337H and R337C, other rationally designed missense mutations targeting a significant salt-bridge interaction that stabilizes the p53 tetramerization domain were studied (i.e., R337D, D352R, and the double-mutation R337D plus D352R). The simulations revealed a destabilizing effect of the pathogenic mutations within the p53 tetramerization domain and highlighted the importance of electrostatic interactions between residues 337 and 352. The transactivation assay, performed in yeast by tuning the expression of wild-type and mutant p53 proteins, revealed that p53 tetramerization mutations could decrease the transactivation potential and alter transactivation specificity, in particular by better tolerating negative features in weak DNA-binding sites. These results establish the effect of naturally occurring variations at positions 337 and 352 on p53’s conformational stability and function.

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.

It might have been believed that elucidation of the atomistic structure of PrP ^Sc would lead to an immediate understanding of the mechanism of prion propagation. However, PrP ^Sc , now known to be a “simple” amyloid, can only template a previously unfolded polypeptide chain. Therefore, PrP ^Sc can easily template the disordered ~90–120 domain of an incoming PrP ^C molecule, but not its ~121–231 folded domain (FD). The FD needs to accommodate into the ~121–230 PrP ^Sc surface, an inert “procrustean bed”. Thus, a mechanism for concerted unfolding/refolding of the FD must exist, with FD unfolding as a key element. To explore how this might happen, we performed thermal unfolding of recombinant bank vole PrP ^C (90–231), a universal PrP ^Sc propagator, tracking changes at the residue level with solution NMR to pinpoint early unfolding propensity. Our data suggest that a key early event is the destabilization of the short β1-β2 assembly and that the segment contiguous to the disordered tail, ~121–140, encompassing β1 and its adjacent coils, is the most likely region to unfold first. Spectroscopic data obtained at higher temperatures suggest that portions of alpha helix α2 are likely the last elements of the FD to unfold and refold into the PrP ^Sc conformation. Molecular Dynamics simulations assisted the interpretation of these changes and suggest separation of α1 from the rest of the FD ensemble. Our data provide a conceivable timeline of the early events in PrP ^Sc -assisted conversion of PrP ^C and should serve as a starting framework to develop a future atomistic model of PrP ^Sc propagation.

The paradigmatic sequence–structure–dynamics–function relation in proteins is currently well established in the scientific community; in particular, a large effort has been made to probe the first connection, indeed providing convincing evidence of its strength and rationalizing it in a quantitative and general framework. In contrast, however, the role of dynamics as a link between structure and function has eluded a similarly clear-cut verification and description. In this work, we propose a pipeline aimed at building a basis for the quantitative characterization of the large-scale dynamics of a set of proteins, starting from the sole knowledge of their native structures. The method hinges on a dynamics-based clusterization, which allows a straightforward comparison with structural and functional protein classifications. The resulting basis set, obtained through the application to a group of related proteins, is shown to reproduce the salient large-scale dynamical features of the dataset. Most interestingly, the basis set is shown to encode the fluctuation patterns of homologous proteins not belonging to the initial dataset, thus highlighting the general applicability of the pipeline used to build it.