Conformational response to ligand binding of TMPRSS2, a protease involved in SARS-CoV-2 infection: Insights through computational modeling.

Thanks to the considerable research which has been undertaken in the last few years to improve our understanding of the biology and mechanism of action of SARS-CoV-2, we know how the virus uses its surface spike protein to infect host cells. The transmembrane prosthesis, serine 2 (TMPRSS2) protein, located on the surface of human cells, recognizes the cleavage site in the spike protein, leading to the release of the fusion peptide and entry of the virus into the host cells. Because of its role, TMPRSS2 has been proposed as a drug target to prevent infection by the virus. In this study, we aim to increase our understanding of TMPRSS2 using long scale microsecond atomistic molecular dynamics simulations, focusing on the conformational changes over time. The comparison between simulations conducted on the protein in the native (apo) and inhibited form (holo), has shown that in the holo form the inhibitor stabilizes the catalytic site and induces rearrangements in the extracellular domain of the protein. In turn, it leads to the formation of a new cavity in the vicinity of the ligand binding pocket that is stable in the microsecond time scale. Given the low specificity of known protease inhibitors, these findings suggest a new potential drug target site that can be used to improve TMPRSS2 specific recognition by newly designed inhibitors.

Distinct dynamical features of plasmodial and human HSP70-HSP110 highlight the divergence in their chaperone-assisted protein folding.

HSP70 and its evolutionarily diverged co-chaperone HSP110, forms an important node in protein folding cascade. How these proteins maintain the aggregation-prone proteome of malaria parasite in functional state remains underexplored, in contrast to its human orthologs. In this study, we have probed into conformational dynamics of plasmodial HSP70 and HSP110 through multiple µs MD-simulations (ATP-state) and compared with their respective human counterparts. Simulations covered sampling of 3.4 and 2.8 µs for HSP70 and HSP110, respectively, for parasite and human orthologs. We provide a comprehensive description of the dynamic behaviors that characterize the systems and also introduce a parameter for quantifying protein rigidity. For HSP70, the interspecies comparison reveals enhanced flexibility in IA and IB subdomain within the conserved NBD, lesser solvent accessibility of the interdomain linker and distinct dynamics of the SBDß of Pf HSP70 in comparison to Hs HSP70. In the case of HSP110, notable contrast in the dynamics of NBD, SBDß and SBDα was observed between parasite and human ortholog. Although HSP70 and HSP110 are members of the same superfamily, we identified specific differences in the subdomain contacts in NBD, linker properties and interdomain movements in their human and parasite orthologs. Our study suggests that differences in conformational dynamics may translate into species-specific differences in the chaperoning activities of HSP70-HSP110 in the parasite and human, respectively. Dynamical features of Pf HSP70-HSP110 may contribute to the maintenance of proteostasis in the parasite during its intracellular survival in the host.

Computational Study of Helicase from SARS-CoV-2 in RNA-Free and Engaged Form.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the causative agent of the pandemic that broke out in 2020 and continues to be the cause of massive global upheaval. Coronaviruses are positive-strand RNA viruses with a genome of ~30 kb. The genome is replicated and transcribed by RNA-dependent RNA polymerase together with accessory factors. One of the latter is the protein helicase (NSP13), which is essential for viral replication. The recently solved helicase structure revealed a tertiary structure composed of five domains. Here, we investigated NSP13 from a structural point of view, comparing its RNA-free form with the RNA-engaged form by using atomistic molecular dynamics (MD) simulations at the microsecond timescale. Structural analyses revealed conformational changes that provide insights into the contribution of the different domains, identifying the residues responsible for domain-domain interactions in both observed forms. The RNA-free system appears to be more flexible than the RNA-engaged form. This result underlies the stabilizing role of the nucleic acid and the functional core role of these domains.

Dominant ARF3 variants disrupt Golgi integrity and cause a neurodevelopmental disorder recapitulated in zebrafish.

Vesicle biogenesis, trafficking and signaling via Endoplasmic reticulum-Golgi network support essential developmental processes and their disruption lead to neurodevelopmental disorders and neurodegeneration. We report that de novo missense variants in ARF3, encoding a small GTPase regulating Golgi dynamics, cause a developmental disease in humans impairing nervous system and skeletal formation. Microcephaly-associated ARF3 variants affect residues within the guanine nucleotide binding pocket and variably perturb protein stability and GTP/GDP binding. Functional analysis demonstrates variably disruptive consequences of ARF3 variants on Golgi morphology, vesicles assembly and trafficking. Disease modeling in zebrafish validates further the dominant behavior of the mutants and their differential impact on brain and body plan formation, recapitulating the variable disease expression. In-depth in vivo analyses traces back impaired neural precursors' proliferation and planar cell polarity-dependent cell movements as the earliest detectable effects. Our findings document a key role of ARF3 in Golgi function and demonstrate its pleiotropic impact on development.

Expanding the molecular spectrum of pathogenic SHOC2 variants underlying Mazzanti syndrome.

We previously molecularly and clinically characterized Mazzanti syndrome, a RASopathy related to Noonan syndrome that is mostly caused by a single recurrent missense variant (c.4A > G, p.Ser2Gly) in SHOC2, which encodes a leucine-rich repeat-containing protein facilitating signal flow through the RAS-mitogen-associated protein kinase (MAPK) pathway. We also documented that the pathogenic p.Ser2Gly substitution causes upregulation of MAPK signaling and constitutive targeting of SHOC2 to the plasma membrane due to the introduction of an N-myristoylation recognition motif. The almost invariant occurrence of the pathogenic c.4A > G missense change in SHOC2 is mirrored by a relatively homogeneous clinical phenotype of Mazzanti syndrome. Here, we provide new data on the clinical spectrum and molecular diversity of this disorder and functionally characterize new pathogenic variants. The clinical phenotype of six unrelated individuals carrying novel disease-causing SHOC2 variants is delineated, and public and newly collected clinical data are utilized to profile the disorder. In silico, in vitro and in vivo characterization of the newly identified variants provides evidence that the consequences of these missense changes on SHOC2 functional behavior differ from what had been observed for the canonical p.Ser2Gly change but converge toward an enhanced activation of the RAS-MAPK pathway. Our findings expand the molecular spectrum of pathogenic SHOC2 variants, provide a more accurate picture of the phenotypic expression associated with variants in this gene and definitively establish a gain-of-function behavior as the mechanism of disease.

Identification of Natural Products as Potential Pharmacological Chaperones for Protein Misfolding Diseases.

Defective protein folding and accumulation of misfolded proteins is associated with neurodegenerative, cardiovascular, secretory, and metabolic disorders. Efforts are being made to identify small-molecule modulators or structural-correctors for conformationally destabilized proteins implicated in various protein aggregation diseases. Using a metastable-reporter-based primary screen, we evaluated pharmacological chaperone activity of a diverse class of natural products. We found that a flavonoid glycoside (C-10, chrysoeriol-7-O-ß-D-glucopyranoside) stabilizes metastable proteins, prevents its aggregation, and remodels the oligomers into protease-sensitive species. Data was corroborated with additional secondary screen with disease-specific pathogenic protein. In vitro and cell-based experiments showed that C-10 inhibits α-synuclein aggregation which is implicated in synucleinopathies-related neurodegeneration. C-10 interferes in its structural transition into ß-sheeted fibrils and mitigates α-synuclein aggregation-associated cytotoxic effects. Computational modeling suggests that C-10 binds to unique sites in α-synuclein which may interfere in its aggregation amplification. These findings open an avenue for comprehensive SAR development for flavonoid glycosides as pharmacological chaperones for metastable and aggregation-prone proteins implicated in protein conformational diseases.

Unsupervised search of low-lying conformers with spectroscopic accuracy: A two-step algorithm rooted into the island model evolutionary algorithm.

The fruitful interplay of high-resolution spectroscopy and quantum chemistry has a long history, especially in the field of small, semi-rigid molecules. However, in recent years, the targets of spectroscopic studies are shifting toward flexible molecules, characterized by a large number of closely spaced energy minima, all contributing to the overall spectrum. Here, artificial intelligence comes into play since it is at the basis of powerful unsupervised techniques for the exploration of soft degrees of freedom. Integration of such algorithms with a two-stage QM/QM' (Quantum Mechanical) exploration/refinement strategy driven by a user-friendly graphical interface is the topic of the present paper. We will address in particular: (i) the performances of different semi-empirical methods for the exploration step and (ii) the comparison between stochastic and meta-heuristic algorithms in achieving a cheap yet complete exploration of the conformational space for medium sized chromophores. As test cases, we choose three amino acids of increasing complexity, whose full conformer enumeration has been reached only very recently. Next, we show that systems in condensed phases can be treated at the same level and with the same efficiency when employing a polarizable continuum description of the solvent. Finally, the challenging issue represented by the vibrational circular dichroism spectra of some rhodium complexes with flexible ligands has been addressed, showing that our fully unsupervised approach leads to remarkable agreement with the experiment.

Computational Spectroscopy in Solution by Integration of Variational and Perturbative Approaches on Top of Clusterized Molecular Dynamics.

Multiscale QM/MM approaches have become the most suitable and effective methods for the investigation of spectroscopic properties of medium- or large-size chromophores in condensed phases. On these grounds, we are developing a novel workflow aimed at improving the generality, reliability, and ease of use of the available tools. In the present paper, we report the latest developments of such an approach with specific reference to a general workplan starting with the addition of acetonitrile to the panel of solvents already available in the General Liquid Optimized Boundary (GLOB) model enforcing nonperiodic boundary conditions (NPBC). Next, the solvatochromic shifts induced by acetonitrile on both rigid (uracil and thymine) and flexible (thyrosine) chromophores have been studied introducing in our software a number of new features ranging from rigid-geometry NPBC molecular dynamics based on the quaternion formalism to a full integration of variational (ONIOM) and perturbative (perturbed matrix method (PMM)) approaches for describing different solute-solvent topologies and local fluctuations, respectively. Finally, thymine and uracil have been studied also in methanol to point out the generality of the computational strategy. While further developments are surely needed, the strengths of our integrated approach even in its present version are demonstrated by the accuracy of the results obtained by an unsupervised approach and coupled to a computational cost strongly reduced with respect to that of conventional QM/MM models without any appreciable accuracy deterioration.

Two-level stochastic search of low-energy conformers for molecular spectroscopy: implementation and validation of MM and QM models.

The search for stationary points in the molecular potential energy surfaces (PES) is a problem of increasing relevance in different fields of molecular sciences especially for large, flexible systems characterized by several large-amplitude internal motions leading to shallow minima with comparable energies and separated by small barriers. After structural biology and medicinal chemistry, also high-resolution molecular spectroscopy, which is the focus of our research activity, is nowadays shifting its attention to this kind of molecular systems. In such circumstances, accurate geometrical structures and relative stabilities of all these minima are a mandatory prerequisite for the vis-à-vis comparison between computed and experimental spectra. This task raises, in turn, the problem of the best compromise between accuracy and feasibility. In our opinion, a promising route is offered by a two-level stochastic search in which a relatively inexpensive MM or QM method is used in the initial search, followed by single point energy evaluation at a higher QM level of a relatively large number of low-energy structures in order to select a final short-list of candidates, whose geometries are fully optimized at the higher QM level. Finally, the relative stabilities and properties of the final short-list of energy minima can be computed by a state-of-the-art QM approach. This strategy defines a general two-level search/three-level evaluation approach, which can be finely tuned in terms of the accuracy of the sought results. Setup of the procedure, interface with a general purpose electronic structure code and validation of the most effective low-level methods for some representative molecular systems (three already well characterized and one new) ended up with a general, robust and user-friendly tool, which can be easily used and extended also by non-specialists to aid experimental spectroscopic studies.

Clinical and functional characterization of a novel RASopathy-causing SHOC2 mutation associated with prenatal-onset hypertrophic cardiomyopathy.

SHOC2 is a scaffold protein mediating RAS-promoted activation of mitogen-activated protein kinase (MAPK) signaling in response to extracellular stimuli. A recurrent activating mutation in SHOC2 (p.Ser2Gly) causes Mazzanti syndrome, a RASopathy characterized by features resembling Noonan syndrome and distinctive ectodermal abnormalities. A second mutation (p.Met173Ile) supposed to cause loss-of-function was more recently identified in two individuals with milder phenotypes. Here, we report on the third RASopathy-causing SHOC2 mutation (c.807_808delinsTT, p.Gln269_His270delinsHisTyr), which was found associated with prenatal-onset hypertrophic cardiomyopathy. Structural analyses indicated a possible impact of the mutation on the relative orientation of the two SHOC2's leucine-rich repeat domains. Functional studies provided evidence of its activating role, revealing enhanced binding of the mutant protein to MRAS and PPP1CB, and increased signaling through the MAPK cascade. Differing from SHOC2 S2G , SHOC2 Q269_H270delinsHY is not constitutively targeted to the plasma membrane. These data document that diverse mechanisms in SHOC2 functional dysregulation converge toward MAPK signaling upregulation.

Smyd2 conformational changes in response to p53 binding: role of the C-terminal domain.

Smyd2 lysine methyltransferase regulates monomethylation of histone and nonhistone lysine residues using S-adenosylmethionine cofactor as the methyl donor. The nonhistone interactors include several tumorigenic targets, including p53. Understanding this interaction would allow the structural principles that underpin Smyd2-mediated p53 methylation to be elucidated. Here, we performed µ-second molecular dynamics (MD) simulations on binary Smyd2-cofactor and ternary Smyd2-cofactor-p53 peptide complexes. We considered both unmethylated and monomethylated p53 peptides (at Lys370 and Lys372). The results indicate that (a) the degree of conformational freedom of the C-terminal domain of Smyd2 is restricted by the presence of the p53 peptide substrate, (b) the Smyd2 C-terminal domain shows distinct dynamic properties when interacting with unmethylated and methylated p53 peptides, and (c) Lys372 methylation confines the p53 peptide conformation, with detectable influence on Lys370 accessibility to the cofactor. These MD results are therefore of relevance for studying the biology of p53 in cancer progression.

Effective yet reliable computation of hyperfine coupling constants in solution by a QM/MM approach: Interplay between electrostatics and non-electrostatic effects.

In this paper, we have extended to the calculation of hyperfine coupling constants, the model recently proposed by some of the present authors [Giovannini et al., J. Chem. Theory Comput. 13, 4854-4870 (2017)] to include Pauli repulsion and dispersion effects in Quantum Mechanical/Molecular Mechanics (QM/MM) approaches. The peculiarity of the proposed approach stands in the fact that repulsion/dispersion contributions are explicitly introduced in the QM Hamiltonian. Therefore, such terms not only enter the evaluation of energetic properties but also propagate to molecular properties and spectra. A novel parametrization of the electrostatic fluctuating charge force field has been developed, thus allowing a quantitative reproduction of reference QM interaction energies. Such a parametrization has been then tested against the prediction of EPR parameters of prototypical nitroxide radicals in aqueous solutions.

Assessment of Multi-Scale Approaches for Computing UV-Vis Spectra in Condensed Phases: Toward an Effective yet Reliable Integration of Variational and Perturbative QM/MM Approaches.

Computational simulation of UV/vis spectra in condensed phases can be performed starting from converged molecular dynamics (MD) simulations and then performing quantum mechanical/molecular mechanical (QM/MM) computations for a statistically significant number of snapshots. However, the need of variational solutions (e.g., ONIOM/EE) for a huge number of snapshots makes unpractical the use of state-of-the-art QM Hamiltonians. On the other hand, the effectivity of perturbative approaches (e.g., perturbed matrix method, PMM) comes at the price of poor convergence for configurations strongly different from the reference one. In this paper we introduce an integrated strategy based on a cluster analysis of the MD snapshots. Next, a representative configuration for each cluster is treated at the ONIOM/EE level, whereas local fluctuations within each cluster are described at the PMM level. Some representative systems (uracil in dimethylformamide and in water and tyrosine zwitterion in water) are analyzed to show the effectivity and flexibility of the proposed strategy.

Interplay between lipid lateral diffusion, dye concentration and membrane permeability unveiled by a combined spectroscopic and computational study of a model lipid bilayer.

Lipid lateral diffusion in membrane bilayers is a fundamental process exploited by cells to enable complex protein structural and dynamic reorganizations. For its importance, lipid mobility in both cellular and model bilayers has been extensively investigated in recent years, especially through the application of time-resolved, fluorescence-based, optical microscopy techniques. However, one caveat of fluorescence techniques is the need to use dye-labeled variants of the lipid of interest, thus potentially perturbing the structural and dynamic properties of the native species. Generally, the effect of the dye/tracer molecule is implicitly assumed to be negligible. Nevertheless, in view of the widespread use of optically modified lipids for studying lipid bilayer dynamics, it is highly desirable to well assess this point. Here, fluorescence correlation spectroscopy (FCS) and molecular dynamics (MD) simulations have been combined together to uncover subtle structural and dynamic effects in DOPC planar membranes enriched with a standard Rhodamine-labeled lipid. Our findings support a non-neutral role of the dye-labeled lipids in diffusion experiments, quantitatively estimating a decrease in lipid mobility of up to 20% with respect to the unlabeled species. Moreover, results highlight the existing interplay between dye concentration, lipid lateral diffusion and membrane permeability, thus suggesting possible implications for future optical microscopy studies of biophysical processes occurring at the membrane level.

Mechanistic insights into metal ions transit through threefold ferritin channel.

BACKGROUND: The mechanism of how the hydrophilic threefold channel (C3) of ferritin nanocages facilitates diffusion of diverse metal ions into the internal cavity remains poorly explored. METHODS: Computational modeling and free energy estimations were carried out on R. catesbeiana H´ ferritin. Transit features and associated energetics for Fe2+, Mg2+, Zn2+ ions through the C3 channel have been examined. RESULTS: We highlight that iron conduction requires the involvement of two Fe2+ ions in the channel. In such doubly occupied configuration, as observed in X-ray structures, Fe2+ is displaced from the internal site (stabilized by D127) at lower energetic cost. Moreover, comparison of Fe2+, Mg2+ and Zn2+ transit features shows that E130 geometric constriction provides not only an electrostatic anchor to the incoming ions but also differentially influence their diffusion kinetics. CONCLUSIONS: Overall, the study provides insights into Fe2+ entry mechanism and characteristic features of metal-protein interactions that influence the metal ions passage. The dynamics data suggest that E130 may act as a metal selectivity gate. This implicates an ion-specific entry mechanism through the channel with the distinct diffusion kinetics being the discriminating factor. GENERAL SIGNIFICANCE: Ferritin nanocages not only act as biological iron reservoirs but also have gained importance in material science as template scaffolds for synthesizing metal nanoparticles. This study provides mechanistic understanding on the conduction of different metal ions through the channel.

Tailor-made computational protocols for precise characterization of small biological building blocks using QM and MM approaches.

Computational modeling involving Quantum Mechanics (QM) and Molecular Mechanics (MM) calculations are widely utilized to unveil the atomic-molecular properties that underpin their inherent characteristic features. The choice over the either of the QM and MM methods or a multiscale composite approach is driven by the target property of interest, and of course, the molecular size. Often, tailor-made schemes need to be devised to match the specific study purpose. Herein, we provide a perspective of these approaches addressing their effectiveness in terms of the delicate balance between the accuracy and computational feasibility. We focus on representative examples to highlight how different approaches can be fruitfully exploited for modeling the conformational landscape, and possibly, the spectroscopic behavior of biochemical molecules, especially amino acids building blocks.

Understanding organellar protein folding capacities and assessing their pharmacological modulation by small molecules.

Dysfunctional organellar protein quality control machinery leads to protein misfolding associated cardiovascular, neurodegenerative, metabolic and secretory disorders. To understand organellar homeostasis, suitable tools are required which can sense changes in their respective protein folding capacity upon exposure to environmental and pharmacological perturbations. Herein, we have assessed protein folding capacity of cellular organelles using a metastable sensor selectively targeted to cytosol, nucleus, mitochondria, endoplasmic reticulum, golgi and peroxisomes. Microscopy and biochemical data revealed that these sensors report both acute and organelle-specific cellular insults. It also provided insights into contrasting refolding capacities of cellular organelles to recover from proteotoxic challenges. Further, we used these metastable sensors to evaluate pharmacological modulation of organellar protein folding capacity by small molecules. We observed pyrazole based scaffolds increased organellar protein folding capacity through upregulation of chaperones, mainly HSP90 and its co-chaperone HOP which coordinate refolding of misfolded/aggregated species. Overall, our data highlights the potential use of organelle-specific metastable sensors to understand protein folding capacity of sub-cellular compartments and assess pharmacological correction of their proteostasis imbalance. This study also provides additional avenue for use of these organelle-specific metastable sensors in drug discovery programs for identification of novel pharmacophores and drug repositioning of promising scaffolds for protein conformational diseases associated with different cellular organelles.