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 serpin plasminogen activator inhibitor 1 (PAI-1) spontaneously undergoes a massive structural change from a metastable, active conformation, with a solvent accessible reactive center loop (RCL), to a stable, inactive or latent conformation in which the RCL has inserted into the central β sheet. Physiologically, conversion to the latent state is regulated by the binding of vitronectin which retards the rate of this latency transition approximately 2-fold. We investigated the effects of vitronectin on the PAI-1 latency transition using all-atom path sampling simulations in explicit solvent. In simulated latency transitions of free PAI-1, the RCL is quite mobile as is the gate, the region that impedes RCL access to the central β sheet. This mobility allows the formation of a transient salt bridge that facilitates the transition, and this finding rationalizes existing mutagenesis results. Vitronectin binding reduces RCL and gate mobility by allosterically rigidifying structural elements over 40 Å away from the binding site thus blocking the transition to the latent conformation. The effects of vitronectin are propagated by a network of dynamically correlated residues including a number of conserved sites that have previously been identified as important for PAI-1 stability. Simulations also revealed a transient pocket populated only in the vitronectin bound state which corresponds to a cryptic drug binding site identified by crystallography. Overall, these results shed new light on regulation of the PAI-1 latency transition by vitronectin and illustrate the potential of path sampling simulations for understanding functional conformational changes in proteins and for facilitating drug discovery.

Prions are self-replicative protein particles lacking nucleic acids. Originally discovered for causing infectious neurodegenerative disorders, they have also been found to play several physiological roles in a variety of species. Functional and pathogenic prions share a common mechanism of replication, characterized by the ability of an amyloid conformer to propagate by inducing the conversion of its physiological, soluble counterpart. In this work, we focus on the propagation of the prion forming domain of HET-s, a physiological fungal prion for which high-resolution structural data are available. Since time-resolved biophysical experiments cannot yield a full reconstruction of prion replication, we resort to computational methods. To overcome the computational limitations of plain Molecular Dynamics (MD) simulations, we adopt a special type of biased dynamics called ratchet-and-pawl MD (rMD). The accuracy of this enhanced path sampling protocol strongly depends on the choice of the collective variable (CV) used to define the biasing force. Since for prion propagation a reliable reaction coordinate (RC) is not yet available, we resort to the recently developed Self-Consistent Path Sampling (SCPS). Indeed, in such an approach the CV where the biasing force is applied is not heuristically postulated but is calculated through an iterative refinement procedure. Our atomistic reconstruction of the HET-s replication shows remarkable similarities with a previously reported mechanism of mammalian PrP Sc propagation obtained with a different computational protocol. Together, these results indicate that the propagation of prions generated by evolutionary distant proteins shares common features. In particular, in both these cases, prions propagate their conformation through a very similar templating mechanism.

Prions are self-replicative protein particles lacking nucleic acids. Originally discovered for causing infectious neurodegenerative disorders, they have also been found to play several physiological roles in a variety of species. Functional and pathogenic prions share a common mechanism of replication, characterized by the ability of an amyloid conformer to propagate by inducing the conversion of its physiological, soluble counterpart. Since timeresolved biophysical experiments are currently unable to provide full reconstruction of the physico-chemical mechanisms responsible for prion replication, one must rely on computer simulations. In this work, we show that a recently developed algorithm called Self-Consistent Path Sampling (SCPS) overcomes the computational limitations of plain MD and provides a viable tool to investigate prion replication processes using state-of-the-art all-atom force fields in explicit solvent. First, we validate the reliability of SCPS simulations by characterizing the folding of a class of small proteins and comparing against the results of plain MD simulations. Next, we use SCPS to investigate the replication of the prion forming domain of HET-s, a physiological fungal prion for which high-resolution structural data are available. Our atomistic reconstruction shows remarkable similarities with a previously reported mechanism of mammalian PrP Sc propagation obtained using a simpler and more approximate path sampling algorithm. Together, these results suggest that the propagation of prions generated by evolutionary distant proteins may share common features. In particular, in both these cases, prions propagate their conformation through a very similar templating mechanism.