Computational approaches to lipid-based nucleic acid delivery systems.

Nucleic acid-based therapies have shown enormous effectiveness as vaccines against the recent COVID19 pandemics and hold great promises in the fight of a broad spectrum of diseases ranging from viral infections to cancer up to genetically transmitted pathologies. Due to their highly degradable polyanionic nature, nucleic acids need to be packed in sophisticate delivery vehicles which compact them up, protect them from early degradation and help delivery them to the right tissue/cells. Lipid-based nanoparticles (LNP) represent, at present, the main solution for nucleic acid delivery. They are made of a mixture of lipids whose key ingredient is an ionizable cationic lipid. Indeed, the interactions between the polyanionic nucleic acids and the ionizable cationic lipids, and their pH-dependent regulation in the life cycle of the nanoparticle, from production to cargo delivery, mostly determine the effectiveness of the therapeutic approach. Notwithstanding the large improvements in the delivery efficiency of LNPs in the last two decades, it is estimated that only a small fraction of the cargo is actually delivered, stimulating further research for the design of more effective LNP formulations. A rationally driven design would profit from the knowledge of the precise molecular structure of these materials, which is however still either missing or characterized by poor spatial resolution. Computational approaches have often been used as a molecular microscope either to enrich the available experimental data and provide a molecular-level picture of the LNPs or even simulate specific processes involving the formation and/or the molecular mechanisms of action of the LNP. Here, I review the recent literature in the field.

pH-Dependent Behavior of Ionizable Cationic Lipids in mRNA-Carrying Lipoplexes Investigated by Molecular Dynamics Simulations.

Lipid-based nanoparticles and lipoplexes containing ionizable lipids are among the most successful nanocarriers for mRNA-based therapies. The molecular structure of these assemblies is still not fully understood, as well as the role played by the ionizable lipids. SAXS experiments have shown that lipoplexes including the ionizable lipid 2-dioleyloxy-N,N-dimethyl-3-aminopropane (DODMA), under specific conditions, have a lamellar structure, where lipid bilayers are separated by mRNA-rich layers, with an overall spacing between 6.5 and 8.0 nm and a complex pH-dependence. Here, the structure and dynamics of these lipoplexes are investigated at varying pH and mRNA concentration using multiscale molecular dynamics simulations. It is observed that the interaction between DODMA and RNA is slightly attractive only at low pH levels, while it becomes effectively repulsive at high and intermediate pH. This results into a pH-dependent relocation of the RNA inside the multilayers, from the lipid head groups at low pH to a more uniform distribution inside the hydrophilic slabs of the multilayers at high pH. It is also observed that at high pH, DODMA lipids shift toward the hydrophobic part of the bilayer, consequently increasing their leaflet-flipping rate, a phenomenon which may ultimately affect the fusion process of the lipoplex with the endosomal membrane.

The Internal Dynamics of Fibrinogen and Its Implications for Coagulation and Adsorption.

Fibrinogen is a serum multi-chain protein which, when activated, aggregates to form fibrin, one of the main components of a blood clot. Fibrinolysis controls blood clot dissolution through the action of the enzyme plasmin, which cleaves fibrin at specific locations. Although the main biochemical factors involved in fibrin formation and lysis have been identified, a clear mechanistic picture of how these processes take place is not available yet. This picture would be instrumental, for example, for the design of improved thrombolytic or anti-haemorrhagic strategies, as well as, materials with improved biocompatibility. Here, we present extensive molecular dynamics simulations of fibrinogen which reveal large bending motions centered at a hinge point in the coiled-coil regions of the molecule. This feature, likely conserved across vertebrates according to our analysis, suggests an explanation for the mechanism of exposure to lysis of the plasmin cleavage sites on fibrinogen coiled-coil region. It also explains the conformational variability of fibrinogen observed during its adsorption on inorganic surfaces and it is supposed to play a major role in the determination of the hydrodynamic properties of fibrinogen. In addition the simulations suggest how the dynamics of the D region of fibrinogen may contribute to the allosteric regulation of the blood coagulation cascade through a dynamic coupling between the a- and b-holes, important for fibrin polymerization, and the integrin binding site P1.

Molecular Dynamics Simulations of the Initial Adsorption Stages of Fibrinogen on Mica and Graphite Surfaces.

Fibrinogen, a blood glycoprotein of vertebrates, plays an essential role in blood clotting by polymerizing into fibrin when activated. Upon adsorption on material surfaces, it also contributes to determine their biocompatibility and has been implicated in the onset of thrombosis and inflammation at medical implants. Here we present the first fully atomistic simulations of the initial stages of the adsorption process of fibrinogen on mica and graphite surfaces. The simulations reveal a weak adsorption on mica that allows frequent desorption and reorientation events. This adsorption is driven by electrostatic interactions between the protein and the silicate surface as well as the counterion layer. Preferred adsorption orientations for the globular regions of the protein are identified. The adsorption on graphite is found to be stronger with fewer reorientation and desorption events and shows the onset of denaturation of the protein.

Effects of ligand binding on the mechanical properties of ankyrin repeat protein gankyrin.

Ankyrin repeat proteins are elastic materials that unfold and refold sequentially, repeat by repeat, under force. Herein we use atomistic molecular dynamics to compare the mechanical properties of the 7-ankyrin-repeat oncoprotein Gankyrin in isolation and in complex with its binding partner S6-C. We show that the bound S6-C greatly increases the resistance of Gankyrin to mechanical stress. The effect is specific to those repeats of Gankyrin directly in contact with S6-C, and the mechanical 'hot spots' of the interaction map to the same repeats as the thermodynamic hot spots. A consequence of stepwise nature of unfolding and the localized nature of ligand binding is that it impacts on all aspects of the protein's mechanical behavior, including the order of repeat unfolding, the diversity of unfolding pathways accessed, the nature of partially unfolded intermediates, the forces required and the work transferred to the system to unfold the whole protein and its parts. Stepwise unfolding thus provides the means to buffer repeat proteins and their binding partners from mechanical stress in the cell. Our results illustrate how ligand binding can control the mechanical response of proteins. The data also point to a cellular mechano-switching mechanism whereby binding between two partner macromolecules is regulated by mechanical stress.

Structural basis of p53 inactivation by cavity-creating cancer mutations and its implications for the development of mutant p53 reactivators.

The cavity-creating p53 cancer mutation Y220C is an ideal paradigm for developing small-molecule drugs based on protein stabilization. Here, we have systematically analyzed the structural and stability effects of all oncogenic Tyr-to-Cys mutations (Y126C, Y163C, Y205C, Y220C, Y234C, and Y236C) in the p53 DNA-binding domain (DBD). They were all highly destabilizing, drastically lowering the melting temperature of the protein by 8-17 °C. In contrast, two non-cancerous mutations, Y103C and Y107C, had only a moderate effect on protein stability. Differential stabilization of the mutants upon treatment with the anticancer agent arsenic trioxide and stibogluconate revealed an interesting proximity effect. Crystallographic studies complemented by MD simulations showed that two of the mutations, Y234C and Y236C, create internal cavities of different size and shape, whereas the others induce unique surface lesions. The mutation-induced pockets in the Y126C and Y205C mutant were, however, relatively small compared with that of the already druggable Y220C mutant. Intriguingly, our structural studies suggest a pronounced plasticity of the mutation-induced pocket in the frequently occurring Y163C mutant, which may be exploited for the development of small-molecule stabilizers. We point out general principles for reactivating thermolabile cancer mutants and highlight special cases where mutant-specific drugs are needed for the pharmacological rescue of p53 function in tumors.

Multiple conformations of full-length p53 detected with single-molecule fluorescence resonance energy transfer.

The tumor suppressor p53 is a member of the emerging class of proteins that have both folded and intrinsically disordered domains, which are a challenge to structural biology. Its N-terminal domain (NTD) is linked to a folded core domain, which has a disordered link to the folded tetramerization domain, which is followed by a disordered C-terminal domain. The quaternary structure of human p53 has been solved by a combination of NMR spectroscopy, electron microscopy, and small-angle X-ray scattering (SAXS), and the NTD ensemble structure has been solved by NMR and SAXS. The murine p53 is reported to have a different quaternary structure, with the N and C termini interacting. Here, we used single-molecule FRET (SM-FRET) and ensemble FRET to investigate the conformational dynamics of the NTD of p53 in isolation and in the context of tetrameric full-length p53 (flp53). Our results showed that the isolated NTD was extended in solution with a strong preference for residues 66-86 forming a polyproline II conformation. The NTD associated weakly with the DNA binding domain of p53, but not the C termini. We detected multiple conformations in flp53 that were likely to result from the interactions of NTD with the DNA binding domain of each monomeric p53. Overall, the SM-FRET results, in addition to corroborating the previous ensemble findings, enabled the identification of the existence of multiple conformations of p53, which are often averaged and neglected in conventional ensemble techniques. Our study exemplifies the usefulness of SM-FRET in exploring the dynamic landscape of multimeric proteins that contain regions of unstructured domains.

Mechanical unfolding of an ankyrin repeat protein.

Ankryin repeat proteins comprise tandem arrays of a 33-residue, predominantly alpha-helical motif that stacks roughly linearly to produce elongated and superhelical structures. They function as scaffolds mediating a diverse range of protein-protein interactions, and some have been proposed to play a role in mechanical signal transduction processes in the cell. Here we use atomic force microscopy and molecular-dynamics simulations to investigate the natural 7-ankyrin repeat protein gankyrin. We find that gankyrin unfolds under force via multiple distinct pathways. The reactions do not proceed in a cooperative manner, nor do they always involve fully stepwise unfolding of one repeat at a time. The peeling away of half an ankyrin repeat, or one or more ankyrin repeats, occurs at low forces; however, intermediate species are formed that are resistant to high forces, and the simulations indicate that in some instances they are stabilized by nonnative interactions. The unfolding of individual ankyrin repeats generates a refolding force, a feature that may be more easily detected in these proteins than in globular proteins because the refolding of a repeat involves a short contraction distance and incurs a low entropic cost. We discuss the origins of the differences between the force- and chemical-induced unfolding pathways of ankyrin repeat proteins, as well as the differences between the mechanics of natural occurring ankyrin repeat proteins and those of designed consensus ankyin repeat and globular proteins.

Data Reweighting in Metadynamics Simulations.

The data collected along a metadynamics simulation can be used to recover information about the underlying unbiased system by means of a reweighting procedure. Here, we analyze the behavior of several reweighting techniques in terms of the quality of the reconstruction of the underlying unbiased free energy landscape in the early stages of the simulation and propose a simple reweighting scheme that we relate to the other techniques. We then show that the free energy landscape reconstructed from reweighted data can be more accurate than the negative bias potential depending on the reweighting technique, the stage of the simulation, and the adoption of well-tempered or standard metadynamics. While none of the tested reweighting techniques from the literature provides the most accurate results in all the analyzed situations, the one proposed here, in addition to helping simplifying the reweighting procedure, converges quickly and precisely to the underlying free energy surface in all the considered cases, thus allowing for an efficient use of limited simulation data.

Targeting Cavity-Creating p53 Cancer Mutations with Small-Molecule Stabilizers: the Y220X Paradigm.

We have previously shown that the thermolabile, cavity-creating p53 cancer mutant Y220C can be reactivated by small-molecule stabilizers. In our ongoing efforts to unearth druggable variants of the p53 mutome, we have now analyzed the effects of other cancer-associated mutations at codon 220 on the structure, stability, and dynamics of the p53 DNA-binding domain (DBD). We found that the oncogenic Y220H, Y220N, and Y220S mutations are also highly destabilizing, suggesting that they are largely unfolded under physiological conditions. A high-resolution crystal structure of the Y220S mutant DBD revealed a mutation-induced surface crevice similar to that of Y220C, whereas the corresponding pocket's accessibility to small molecules was blocked in the structure of the Y220H mutant. Accordingly, a series of carbazole-based small molecules, designed for stabilizing the Y220C mutant, also bound to and stabilized the folded state of the Y220S mutant, albeit with varying affinities due to structural differences in the binding pocket of the two mutants. Some of the compounds also bound to and stabilized the Y220N mutant, but not the Y220H mutant. Our data validate the Y220S and Y220N mutants as druggable targets and provide a framework for the design of Y220S or Y220N-specific compounds as well as compounds with dual Y220C/Y220S specificity for use in personalized cancer therapy.

High temperature unfolding simulations of the TRPZ1 peptide.

We report high temperature molecular dynamics simulations of the unfolding of the TRPZ1 peptide using an explicit model for the solvent. The system has been simulated for a total of 6 mus with 100-ns minimal continuous stretches of trajectory. The populated states along the simulations are identified by monitoring multiple observables, probing both the structure and the flexibility of the conformations. Several unfolding and refolding transition pathways are sampled and analyzed. The unfolding process of the peptide occurs in two steps because of the accumulation of a metastable on-pathway intermediate state stabilized by two native backbone hydrogen bonds assisted by nonnative hydrophobic interactions between the tryptophan side chains. Analysis of the un/folding kinetics and classical commitment probability calculations on the conformations extracted from the transition pathways show that the rate-limiting step for unfolding is the disruption of the ordered native hydrophobic packing (Trp-zip motif) leading from the native to the intermediate state. But, the speed of the folding process is mainly determined by the transition from the completely unfolded state to the intermediate and specifically by the closure of the hairpin loop driven by formation of two native backbone hydrogen bonds and hydrophobic contacts between tryptophan residues. The temperature dependence of the unfolding time provides an estimate of the unfolding activation enthalpy that is in agreement with experiments. The unfolding time extrapolated to room temperature is in agreement with the experimental data as well, thus providing a further validation to the analysis reported here.

Fluorescence resonance energy transfer analysis of the folding pathway of Engrailed Homeodomain.

The Engrailed Homeodomain folds on the microsecond time scale via an intermediate that is experimentally well characterised using structural Engrailed-Homeodomain mimics. Here, we analysed directly the changes in distance between key residues during the kinetics of unfolding and at equilibrium using fluorescence resonance energy transfer (FRET). Trp was the donor and 5-(((acetylamino)ethyl)amino) naphthalene-1-sulphate, the acceptor, substituted in positions that caused little change in stability. Distances calculated for the native state were in good agreement with those derived from the NMR structure. The distances between the N- and C-termini of Helix I and of Helix III increased, then decreased and finally increased again with increasing GdmCl concentration on equilibrium denaturation. This behaviour implied that there was a folding intermediate on the folding pathway and that this intermediate was populated at low concentrations of GdmCl concentration ( approximately 1 M). We analysed the changes in distance during temperature-jump relaxation kinetics, using a qualitative and very conservative procedure that drew conclusions only when changes in fluorescence of mutants containing either the donor or the acceptor alone would not obscure the change in the FRET signal when both donor and acceptor were present. The distance changes obtained under equilibrium and kinetic measurements were self-consistent and also consistent with the known high-resolution structures of the mimics of the folding intermediates. We showed that for analysing distances in disordered ensembles, it is important to use FRET probes with a critical distance close to the average separation in the ensemble. Otherwise, average distances could be over or underestimated.

Wordom: a program for efficient analysis of molecular dynamics simulations.

Wordom is a versatile program for manipulation of molecular dynamics trajectories and efficient analysis of simulations. Original tools in Wordom include a procedure to evaluate significance of sampling for principal component analysis as well as modules for clustering multiple conformations and evaluation of order parameters for folding and aggregation. The program was developed with special emphasis on user-friendliness, effortless addition of new modules and efficient handling of large sets of trajectories.

Poly-sarcosine and Poly(Ethylene-Glycol) Interactions with Proteins Investigated Using Molecular Dynamics Simulations.

Nanoparticles coated with hydrophilic polymers often show a reduction in unspecific interactions with the biological environment, which improves their biocompatibility. The molecular determinants of this reduction are not very well understood yet, and their knowledge may help improving nanoparticle design. Here we address, using molecular dynamics simulations, the interactions of human serum albumin, the most abundant serum protein, with two promising hydrophilic polymers used for the coating of therapeutic nanoparticles, poly(ethylene-glycol) and poly-sarcosine. By simulating the protein immersed in a polymer-water mixture, we show that the two polymers have a very similar affinity for the protein surface, both in terms of the amount of polymer adsorbed and also in terms of the type of amino acids mainly involved in the interactions. We further analyze the kinetics of adsorption and how it affects the polymer conformations. Minor differences between the polymers are observed in the thickness of the adsorption layer, that are related to the different degree of flexibility of the two molecules. In comparison poly-alanine, an isomer of poly-sarcosine known to self-aggregate and induce protein aggregation, shows a significantly larger affinity for the protein surface than PEG and PSar, which we show to be related not to a different patterns of interactions with the protein surface, but to the different way the polymer interacts with water.

Estimation of folding probabilities and phi values from molecular dynamics simulations of reversible Peptide folding.

Molecular dynamics simulations with an implicit model of the solvent have allowed to investigate the reversible folding of structured peptides. For a 20-residue antiparallel beta-sheet peptide, the simulation results have revealed multiple folding pathways. Moreover, the conformational heterogeneity of the denatured state has been shown to originate from high enthalpy, high entropy basins with fluctuating non-native secondary structure, as well as low enthalpy, low entropy traps. An efficient and simple approach to estimate folding probabilities from molecular dynamics simulations has allowed to isolate conformations in the transition state ensemble and to evaluate Phi values, i.e., the effects of mutations on the folding kinetics and thermodynamic stability. These molecular dynamics studies have provided evidence that, if interpreted by neglecting the non-native interactions, Phi values overestimate the amount of native-like structure in the transition state.

Protein corona composition of poly(ethylene glycol)- and poly(phosphoester)-coated nanoparticles correlates strongly with the amino acid composition of the protein surface.

Extensive molecular dynamics simulations reveal that the interactions between proteins and poly(ethylene glycol) (PEG) can be described in terms of the surface composition of the proteins. PEG molecules accumulate around non-polar residues while avoiding the polar ones. A solvent-accessible-surface-area model of protein adsorption accurately fits a large set of data on the composition of the protein corona of poly(ethylene glycol)- and poly(phosphoester)-coated nanoparticles recently obtained by label-free proteomic mass spectrometry.

Unfolding transition state and intermediates of the tumor suppressor p16INK4a investigated by molecular dynamics simulations.

The ankyrin repeat is one of the most common protein motifs and is involved in protein-protein interactions. It consists of 33 residues that assume a beta-hairpin helix-loop-helix fold. Mutagenesis and kinetic experiments (Phi-value analysis of the folding transition state) have shown that the tumor suppressor p16(INK4a), a four-repeat protein, unfolds sequentially starting from the two N-terminal repeats. Here, the flexibility of p16(INK4a) at room temperature and its unfolding mechanism at high temperature have been investigated by multiple molecular dynamics runs in explicit water for a total simulation time of 0.65 micros. The transition state ensemble (TSE) of p16(INK4a) was identified by monitoring both the deviation from the experimental Phi values and sudden conformational changes along the unfolding trajectories. Conformations in the TSE have a mainly unstructured second repeat whereas the other repeats are almost completely folded. A rigid-body displacement of the first repeat involving both a rotation and translation is observed in all molecular dynamics simulations at high temperature. The Trp(15), Pro(75), and Ala(76) side-chains are more buried in the TSE than the native state. The sequential unfolding starting at the second repeat is in agreement with the mutagenesis studies whereas the displacement of the first repeat and the presence of nonnative interactions at the TSE are simulation results which supplement the experimental data. Furthermore, the unfolding trajectories reveal the presence of two on-pathway intermediates with partial alpha-helical structure. Finally, on the basis of the available experimental and simulation results we suggest that in modular proteins the shift of the folding TSE toward the native structure upon reduction of the number of tandem repeats is consistent with the Hammond effect.

The intracellular antibody capture technology (IACT): towards a consensus sequence for intracellular antibodies.

We describe the application of an intracellular antibody capture technology (IACT) as a generic in vivo selection procedure for isolating intracellular antibodies or ICAbs. IACT was applied to the de novo selection of functional ICAbs against the microtubule-associated protein TAU, found in neurofibrillary lesions of Alzheimer's disease brains. A panel of 17 different ICAbs was created which bind TAU inside cells and the epitopes recognized by the selected ICAbs have been determined by an in vivo epitope mapping procedure. Finally, sequence analysis showed that the IACT-derived ICAbs are characterized by a common signature of conserved amino acid residues, suggesting that the IACT naturally selects a sort of "captured consensus sequence" for intracellular antibodies. The development of IACT, together with the possibility of scaling up in a high throughput and automated format, makes IACT a new enabling tool for target validation in functional genomics and global proteomics.

Simulations and experiments in protein folding.

The interplay between simulations and experiments of protein folding has largely contributed to the elucidation of many important aspects of the phenomenon. In this chapter, I briefly describe the experiments which provide information on the kinetics of the protein folding process, and help to characterize the folding transition state. Then, I show how to probe the kinetics of protein folding using molecular dynamics simulations, how to compare the simulations with the experiments and how to help and rationalize the latter, ultimately offering a molecular picture of the process. After the production of suitable molecular dynamics simulation data in the form of trajectories, the procedure involves sequentially the identification of the stable states of the protein, the identification of the transition pathways connecting the stable states, the identification of the transition state conformations, comparison with experimental results, and finally, the identification of the molecular determinants or reaction coordinates of the folding process, that is, the features that clearly help distinguishing the transition state from the stable states.

Combination of Markov state models and kinetic networks for the analysis of molecular dynamics simulations of peptide folding.

Atomistic molecular dynamics simulations of the TZ1 beta-hairpin peptide have been carried out using an implicit model for the solvent. The trajectories have been analyzed using a Markov state model defined on the projections along two significant observables and a kinetic network approach. The Markov state model allowed for an unbiased identification of the metastable states of the system, and provided the basis for commitment probability calculations performed on the kinetic network. The kinetic network analysis served to extract the main transition state for folding of the peptide and to validate the results from the Markov state analysis. The combination of the two techniques allowed for a consistent and concise characterization of the dynamics of the peptide. The slowest relaxation process identified is the exchange between variably folded and denatured species, and the second slowest process is the exchange between two different subsets of the denatured state which could not be otherwise identified by simple inspection of the projected trajectory. The third slowest process is the exchange between a fully native and a partially folded intermediate state characterized by a native turn with a proximal backbone H-bond, and frayed side-chain packing and termini. The transition state for the main folding reaction is similar to the intermediate state, although a more native like side-chain packing is observed.