Giulia Rossetti

Structural facets of disease-linked human prion protein mutants: A molecular dynamic study

Prion propagation in transmissible spongiform encephalopathies involves the conversion of the cellular prion protein, PrPC, into the pathogenic conformer PrPSc. Human familial forms of the disease are linked to specific mutations in the PrP gene, PRNP, and include Gerstmann‐Sträussler‐Scheinker syndrome (GSS), familial Creutzfeldt‐Jakob disease (fCJD), and fatal familial insomnia. To gain insights into the molecular basis of these disorders, we performed 200 ns of classical molecular dynamic simulations in aqueous solution on wild type (WT) human PrP (HuPrP), and on three HuPrP variants located in the globular HuPrP domain: two pathological mutations, HuPrP(Q212P) and HuPrP(E200K), linked to GSS and to fCJD respectively, and one protective polymorphism, HuPrP(E219K) (total time‐scale simulated 800 ns). A comparison between the predicted structural determinants of WT HuPrP and HuPrP(E200K) with their NMR structures established the accuracy of the methods used. Strikingly, the analyzed disease‐linked variants produced their major effect on the α2‐α3 region and the β2‐α2 loop, regardless of the mutation position. The conformational change of the latter might affect the interactions with cellular partners in the fibrillation process. The protocol proposed here represents a powerful approach for reproducing the structural effects of genetic mutations located in the globular domain of HuPrP, such as the GSS‐related HuPrP(Q212P) and the protective polymorphism HuPrP(E219K). Proteins 2010.

The structural impact of DNA mismatches

The structure and dynamics of all the transversion and transition mismatches in three different DNA environments have been characterized by molecular dynamics simulations and NMR spectroscopy. We found that the presence of mismatches produced significant local structural alterations, especially in the case of purine transversions. Mismatched pairs often show promiscuous hydrogen bonding patterns, which interchange among each other in the nanosecond time scale. This therefore defines flexible base pairs, where breathing is frequent, and where distortions in helical parameters are strong, resulting in significant alterations in groove dimension. Even if the DNA structure is plastic enough to absorb the structural impact of the mismatch, local structural changes can be propagated far from the mismatch site, following the expected through-backbone and a previously unknown through-space mechanism. The structural changes related to the presence of mismatches help to understand the different susceptibility of mismatches to the action of repairing proteins.

Molecular basis for structural heterogeneity of an intrinsically disordered protein bound to a partner by combined ESI-IM-MS and modeling

AbstractIntrinsically disordered proteins (IDPs) form biologically active complexes that can retain a high degree of conformational disorder, escaping structural characterization by conventional approaches. An example is offered by the complex between the intrinsically disordered NTAIL domain and the phosphoprotein X domain (PXD) from measles virus (MeV). Here, distinct conformers of the complex are detected by electrospray ionization-mass spectrometry (ESI-MS) and ion mobility (IM) techniques yielding estimates for the solvent-accessible surface area (SASA) in solution and the average collision cross-section (CCS) in the gas phase. Computational modeling of the complex in solution, based on experimental constraints, provides atomic-resolution structural models featuring different levels of compactness. The resulting models indicate high structural heterogeneity. The intermolecular interactions are predominantly hydrophobic, not only in the ordered core of the complex, but also in the dynamic, disordered regions. Electrostatic interactions become involved in the more compact states. This system represents an illustrative example of a hydrophobic complex that could be directly detected in the gas phase by native mass spectrometry. This work represents the first attempt to modeling the entire NTAIL domain bound to PXD at atomic resolution. Graphical Abstractᅟ

Docking Ligands on Protein Surfaces: The Case Study of Prion Protein.

Molecular docking of ligands targeting proteins undergoing fibrillization in neurodegenerative diseases is difficult because of the lack of deep binding sites. Here we extend standard docking methods with free energy simulations in explicit solvent to address this issue in the context of the prion protein surface. We focus on a specific ligand (2-pyrrolidin-1-yl-N-[4-[4-(2-pyrrolidin-1-yl-acetylamino)-benzyl]-phenyl]-acetamide), which binds to the structured part of the protein as shown by NMR (Kuwata, K. et al. Proc Natl Acad Sci U.S.A. 2007, 104, 11921-11926). The calculated free energy of dissociation (7.8 ± 0.9 kcal/mol) is in good agreement with the value derived by the experimental dissociation constant (Kd = 3.9 μM, corresponding to ΔG(0) = -7.5 kcal/mol). Several binding poses are predicted, including the one reported previously. Our prediction is fully consistent with the presence of multiple binding sites, emerging from NMR measurements. Our molecular simulation-based approach emerges, therefore, as a useful tool to predict poses and affinities of ligand binding to protein surfaces.

The conserved macrodomains of the non-structural proteins of Chikungunya virus and other pathogenic positive strand RNA viruses function as mono-ADP-ribosylhydrolases

Human pathogenic positive single strand RNA ((+)ssRNA) viruses, including Chikungunya virus, pose severe health problems as for many neither efficient vaccines nor therapeutic strategies exist. To interfere with propagation, viral enzymatic activities are considered potential targets. Here we addressed the function of the viral macrodomains, conserved folds of non-structural proteins of many (+)ssRNA viruses. Macrodomains are closely associated with ADP-ribose function and metabolism. ADP-ribosylation is a post-translational modification controlling various cellular processes, including DNA repair, transcription and stress response. We found that the viral macrodomains possess broad hydrolase activity towards mono-ADP-ribosylated substrates of the mono-ADP-ribosyltransferases ARTD7, ARTD8 and ARTD10 (aka PARP15, PARP14 and PARP10, respectively), reverting this post-translational modification both in vitro and in cells. In contrast, the viral macrodomains possess only weak activity towards poly-ADP-ribose chains synthesized by ARTD1 (aka PARP1). Unlike poly-ADP-ribosylglycohydrolase, which hydrolyzes poly-ADP-ribose chains to individual ADP-ribose units but cannot cleave the amino acid side chain - ADP-ribose bond, the different viral macrodomains release poly-ADP-ribose chains with distinct efficiency. Mutational and structural analyses identified key amino acids for hydrolase activity of the Chikungunya viral macrodomain. Moreover, ARTD8 and ARTD10 are induced by innate immune mechanisms, suggesting that the control of mono-ADP-ribosylation is part of a host-pathogen conflict.

Copper Binding to the N-Terminally Acetylated, Naturally Occurring Form of Alpha-Synuclein Induces Local Helical Folding

Growing evidence supports a link between brain copper homeostasis, the formation of alpha-synuclein (AS)-copper complexes, and the development of Parkinson disease (PD). Recently it was demonstrated that the physiological form of AS is N-terminally acetylated (AcAS). Here we used NMR spectroscopy to structurally characterize the interaction between Cu(I) and AcAS. We found that the formation of an AcAS-Cu(I) complex at the N-terminal region stabilizes local conformations with α-helical secondary structure and restricted motility. Our work provides new evidence into the metallo-biology of PD and opens new lines of research as the formation of AcAS-Cu(I) complex might impact on AcAS membrane binding and aggregation.

Conformations of the Huntingtin N‐term in aqueous solution from atomistic simulations

The first 17 amino acids of Huntingtin protein (N17) play a crucial role in the proteins aggregation. Here we predict its free energy landscape in aqueous solution by using bias exchange metadynamics. All our findings are consistent with experimental data. N17 populates four main kinetic basins, which interconvert on the microsecond time‐scale. The most populated basin (about 75%) is a random coil, with an extended flat exposed hydrophobic surface. This might create a hydrophobic seed promoting Huntingtin aggregation. The other main populated basins contain helical conformations, which could facilitate N17 binding on its cellular targets.

Environmental and genetic factors support the dissociation between α-synuclein aggregation and toxicity.

Significance Many neurodegenerative diseases are characterized by the abnormal accumulation of aggregated proteins in the brain. In Parkinson’s disease and related disorders, this process involves the accumulation of α-synuclein (aSyn). Thus, understanding the relationship between aSyn aggregation and pathological conditions is essential for the development of novel and efficient therapies against these disorders. Here, we studied the effects that different aSyn species have on neurons using a combination of neurodegeneration-associated factors: the H50Q aSyn mutant and the presence of copper. Importantly, we demonstrate that exogenous aSyn promotes toxicity and inclusion formation, and that these effects are inversely correlated. Our data shed light onto the pathological mechanisms associated with aSyn aggregation, forming the foundation for future therapeutic strategies. Synucleinopathies are a group of progressive disorders characterized by the abnormal aggregation and accumulation of α-synuclein (aSyn), an abundant neuronal protein that can adopt different conformations and biological properties. Recently, aSyn pathology was shown to spread between neurons in a prion-like manner. Proteins like aSyn that exhibit self-propagating capacity appear to be able to adopt different stable conformational states, known as protein strains, which can be modulated both by environmental and by protein-intrinsic factors. Here, we analyzed these factors and found that the unique combination of the neurodegeneration-related metal copper and the pathological H50Q aSyn mutation induces a significant alteration in the aggregation properties of aSyn. We compared the aggregation of WT and H50Q aSyn with and without copper, and assessed the effects of the resultant protein species when applied to primary neuronal cultures. The presence of copper induces the formation of structurally different and less-damaging aSyn aggregates. Interestingly, these aggregates exhibit a stronger capacity to induce aSyn inclusion formation in recipient cells, which demonstrates that the structural features of aSyn species determine their effect in neuronal cells and supports a lack of correlation between toxicity and inclusion formation. In total, our study provides strong support in favor of the hypothesis that protein aggregation is not a primary cause of cytotoxicity.

Conformational effects in protein electrospray-ionization mass spectrometry.

Electrospray-ionization mass spectrometry (ESI-MS) is a key tool of structural biology, complementing the information delivered by conventional biochemical and biophysical methods. Yet, the mechanism behind the conformational effects in protein ESI-MS is an object of debate. Two parameters-solvent-accessible surface area (As) and apparent gas-phase basicity (GBapp)-are thought to play a role in controlling the extent of protein ionization during ESI-MS experiments. This review focuses on recent experimental and theoretical investigations concerning the influence of these parameters on ESI-MS results and the structural information that can be derived. The available evidence supports a unified model for the ionization mechanism of folded and unfolded proteins. These data indicate that charge-state distribution (CSD) analysis can provide valuable structural information on normally folded, as well as disordered structures.

Developing predictive rules for coordination geometry from visible circular dichroism of copper(II) and nickel(II) ions in histidine and amide main-chain complexes.

Circular dichroism (CD) spectroscopy in the visible region (vis‐CD) is a powerful technique to study metal–protein interactions. It can resolve individual d–d electronic transitions as separate bands and is particularly sensitive to the chiral environment of the transition metals. Modern quantum chemical methods enable CD spectra calculations from which, along with direct comparison with the experimental CD data, the conformations and the stereochemistry of the metal–protein complexes can be assigned. However, a clear understanding of the observed spectra and the molecular configuration is largely lacking. In this study, we compare the experimental and computed vis‐CD spectra of Cu2+‐loaded model peptides in square‐planar complexes. We find that the spectra can readily discriminate the coordination pattern of Cu2+ bound exclusively to main‐chain amides from that involving both main‐chain amides and a side‐chain (i.e. histidine side‐chain). Based on the results, we develop a set of empirical rules that relates the appearance of particular vis‐CD spectral features to the conformation of the complex. These rules can be used to gain insight into coordination geometries of other Cu2+–or Ni2+–protein complexes.