Kinetics of radiation-induced DNA double-strand breaks through coarse-grained simulations.

Double-strand breaks (DSBs), i.e., the covalent cut of the DNA backbone over both strands, are a detrimental outcome of cell irradiation, bearing chromosomal aberrations and leading to cell apoptosis. In the early stages of the evolution of a DSB, the disruption of the residual interactions between the DNA moieties drives the fracture of the helical layout; in spite of its biological significance, the details of this process are still largely uncertain. Here, we address the mechanical rupture of DNA by DSBs via coarse-grained molecular dynamics simulations: the setup involves a 3855-bp DNA filament and diverse DSB motifs, i.e., within a range of distances between strand breaks (or DSB distance). By employing a coarse-grained model of DNA, we access the molecular details and characteristic timescales of the rupturing process. A sequence-nonspecific, linear correlation is observed between the DSB distance and the internal energy contribution to the disruption of the residual (Watson-Crick and stacking) contacts between DNA moieties, which is seemingly driven by an abrupt, cooperative process. Moreover, we infer an exponential dependence of the characteristic rupture times on the DSB distances, which we associate to an Arrhenius-like law of thermally-activated processes. This work lays the foundations of a detailed, mechanistic assessment of DSBs in silico as a benchmark to both numerical simulations and data from single-molecule experiments.

Gamma-Hemolysin Components: Computational Strategies for LukF-Hlg2 Dimer Reconstruction on a Model Membrane.

The gamma-hemolysin protein is one of the most common pore-forming toxins expressed by the pathogenic bacterium Staphylococcus aureus. The toxin is used by the pathogen to escape the immune system of the host organism, by assembling into octameric transmembrane pores on the surface of the target immune cell and leading to its death by leakage or apoptosis. Despite the high potential risks associated with Staphylococcus aureus infections and the urgent need for new treatments, several aspects of the pore-formation process from gamma-hemolysin are still unclear. These include the identification of the interactions between the individual monomers that lead to the formation of a dimer on the cell membrane, which represents the unit for further oligomerization. Here, we employed a combination of all-atom explicit solvent molecular dynamics simulations and protein-protein docking to determine the stabilizing contacts that guide the formation of a functional dimer. The simulations and the molecular modeling reveal the importance of the flexibility of specific protein domains, in particular the N-terminus, to drive the formation of the correct dimerization interface through functional contacts between the monomers. The results obtained are compared with the experimental data available in the literature.

A Comparative Molecular Dynamics Study of Selected Point Mutations in the Shwachman-Bodian-Diamond Syndrome Protein SBDS.

The Shwachman-Diamond Syndrome (SDS) is an autosomal recessive disease whose majority of patients display mutations in a ribosome assembly protein named Shwachman-Bodian-Diamond Syndrome protein (SBDS). A specific therapy for treating this rare disease is missing, due to the lack of knowledge of the molecular mechanisms responsible for its pathogenesis. Starting from the observation that SBDS single-point mutations, localized in different domains of the proteins, are responsible for an SDS phenotype, we carried out the first comparative Molecular Dynamics simulations on three SBDS mutants, namely R19Q, R126T and I212T. The obtained 450-ns long trajectories were compared with those returned by both the open and closed forms of wild type SBDS and strongly indicated that two distinct conformations (open and closed) are both necessary for the proper SBDS function, in full agreement with recent experimental observations. Our study supports the hypothesis that the SBDS function is governed by an allosteric mechanism involving domains I and III and provides new insights into SDS pathogenesis, thus offering a possible starting point for a specific therapeutic option.

Comparative Molecular Dynamics Investigation of the Electromotile Hearing Protein Prestin.

The mammalian protein prestin is expressed in the lateral membrane wall of the cochlear hair outer cells and is responsible for the electromotile response of the basolateral membrane, following hyperpolarisation or depolarisation of the cells. Its impairment marks the onset of severe diseases, like non-syndromic deafness. Several studies have pointed out possible key roles of residues located in the Transmembrane Domain (TMD) that differentiate mammalian prestins as incomplete transporters from the other proteins belonging to the same solute-carrier (SLC) superfamily, which are classified as complete transporters. Here, we exploit the homology of a prototypical incomplete transporter (rat prestin, rPres) and a complete transporter (zebrafish prestin, zPres) with target structures in the outward open and inward open conformations. The resulting models are then embedded in a model membrane and investigated via a rigorous molecular dynamics simulation protocol. The resulting trajectories are analyzed to obtain quantitative descriptors of the equilibration phase and to assess a structural comparison between proteins in different states, and between different proteins in the same state. Our study clearly identifies a network of key residues at the interface between the gate and the core domains of prestin that might be responsible for the conformational change observed in complete transporters and hindered in incomplete transporters. In addition, we study the pathway of Cl- ions in the presence of an applied electric field towards their putative binding site in the gate domain. Based on our simulations, we propose a tilt and shift mechanism of the helices surrounding the ion binding cavity as the working principle of the reported conformational changes in complete transporters.

Theoretical insights on acceptor-donor dyads for organic photovoltaics.

The field of organic photovoltaics has witnessed a steady growth in the last few decades and a recent renewal with the blossoming of single-material organic solar cells (SMOSCs). However, due to the intrinsic complexity of these devices (both in terms of their size and of the condensed phases involved), computational approaches to accurately predict their geometrical and electronic structure and to link their microscopic properties to the observed macroscopic behaviour are still lacking. In this work, we have focused on the rationalization of transport dynamics and we have set up a computational approach that makes a combined use of classical simulations and Density Functional Theory with the aim of disclosing the most relevant electronic and structural features of dyads used for SMOSC applications. As a prototype dyad, we have considered a molecule that consists in a dithiafulvalene-functionalized diketopyrrolopyrrole (DPP), acting as an electron donor, covalently linked to a fulleropyrrolidine (Ful), the electron acceptor. Our results, beside a quantitative agreement with experiments, show that the overall observed mobilities result from the competing packing mechanisms of the constituting units within the dyad both in the case of crystalline and amorphous phases. As a consequence, not all stable polymorphs have the same efficiency in transporting holes or electrons which often results in a highly directional carrier transport that is not, in general, a desirable feature for polycrystalline thin-films. The present work, linking microscopic packing to observed transport, thus opens the route for the in silico design of new dyads with enhanced and controlled structural and electronic features.

Effects of Membrane and Biological Target on the Structural and Allosteric Properties of Recoverin: A Computational Approach.

Recoverin (Rec) is a prototypical calcium sensor protein primarily expressed in the vertebrate retina. The binding of two Ca2+ ions to the functional EF-hand motifs induces the extrusion of a myristoyl group that increases the affinity of Rec for the membrane and leads to the formation of a complex with rhodopsin kinase (GRK1). Here, unbiased all-atom molecular dynamics simulations were performed to monitor the spontaneous insertion of the myristoyl group into a model multicomponent biological membrane for both isolated Rec and for its complex with a peptide from the GRK1 target. It was found that the functional membrane anchoring of the myristoyl group is triggered by persistent electrostatic protein-membrane interactions. In particular, salt bridges between Arg43, Arg46 and polar heads of phosphatidylserine lipids are necessary to enhance the myristoyl hydrophobic packing in the Rec-GRK1 assembly. The long-distance communication between Ca2+-binding EF-hands and residues at the interface with GRK1 is significantly influenced by the presence of the membrane, which leads to dramatic changes in the connectivity of amino acids mediating the highest number of persistent interactions (hubs). In conclusion, specific membrane composition and allosteric interactions are both necessary for the correct assembly and dynamics of functional Rec-GRK1 complex.

Comparative molecular dynamics study of neuromyelitis optica-immunoglobulin G binding to aquaporin-4 extracellular domains.

Neuromyelitis optica (NMO) is an inflammatory demyelinating disease of the central nervous system in which most patients have serum autoantibodies (called NMO-IgG) that bind to astrocyte water channel aquaporin-4 (AQP4). A potential therapeutic strategy in NMO is to block the interaction of NMO-IgG with AQP4. Building on recent observation that some single-point and compound mutations of the AQP4 extracellular loop C prevent NMO-IgG binding, we carried out comparative Molecular Dynamics (MD) investigations on three AQP4 mutants, TP137-138AA, N153Q and V150G, whose 295-ns long trajectories were compared to that of wild type human AQP4. A robust conclusion of our modeling is that loop C mutations affect the conformation of neighboring extracellular loop A, thereby interfering with NMO-IgG binding. Analysis of individual mutations suggested specific hydrogen bonding and other molecular interactions involved in AQP4-IgG binding to AQP4.

Challenging AQP4 druggability for NMO-IgG antibody binding using molecular dynamics and molecular interaction fields.

Neuromyelitis optica (NMO) is a multiple sclerosis-like immunopathology disease affecting optic nerves and the spinal cord. Its pathological hallmark is the deposition of a typical immunoglobulin, called NMO-IgG, against the water channel Aquaporin-4 (AQP4). Preventing NMO-IgG binding would represent a valuable molecular strategy for a focused NMO therapy. The recent observation that aspartate in position 69 (D69) is determinant for the formation of NMO-IgG epitopes prompted us to carry out intensive Molecular Dynamics (MD) studies on a number of single-point AQP4 mutants. Here, we report a domino effect originating from the point mutation at position 69: we find that the side chain of T62 is reoriented far from its expected position leaning on the lumen of the pore. More importantly, the strength of the H-bond interaction between L53 and T56, at the basis of the loop A, is substantially weakened. These events represent important pieces of a clear-cut mechanistic rationale behind the failure of the NMO-IgG binding, while the water channel function as well as the propensity to aggregate into OAPs remains unaltered. The molecular interaction fields (MIF)-based analysis of cavities complemented MD findings indicating a putative binding site comprising the same residues determining epitope reorganization. In this respect, docking studies unveiled an intriguing perspective to address the future design of small drug-like compounds against NMO. In agreement with recent experimental observations, the present study is the first computational attempt to elucidate NMO-IgG binding at the molecular level, as well as a first effort toward a less elusive AQP4 druggability.

Human Aquaporin-4 and Molecular Modeling: Historical Perspective and View to the Future.

Among the different aquaporins (AQPs), human aquaporin-4 (hAQP4) has attracted the greatest interest in recent years as a new promising therapeutic target. Such a membrane protein is, in fact, involved in a multiple sclerosis-like immunopathology called Neuromyelitis Optica (NMO) and in several disorders resulting from imbalanced water homeostasis such as deafness and cerebral edema. The gap of knowledge in its functioning and dynamics at the atomistic level of detail has hindered the development of rational strategies for designing hAQP4 modulators. The application, lately, of molecular modeling has proved able to fill this gap providing a breeding ground to rationally address compounds targeting hAQP4. In this review, we give an overview of the important advances obtained in this field through the application of Molecular Dynamics (MD) and other complementary modeling techniques. The case studies presented herein are discussed with the aim of providing important clues for computational chemists and biophysicists interested in this field and looking for new challenges.

Identification of a point mutation impairing the binding between aquaporin-4 and neuromyelitis optica autoantibodies.

Neuromyelitis optica (NMO) is characterized by the presence of pathogenic autoantibodies (NMO-IgGs) against supra-molecular assemblies of aquaporin-4 (AQP4), known as orthogonal array of particles (OAPs). NMO-IgGs have a polyclonal origin and recognize different conformational epitopes involving extracellular AQP4 loops A, C, and E. Here we hypothesize a pivotal role for AQP4 transmembrane regions (TMs) in epitope assembly. On the basis of multialignment analysis, mutagenesis, NMO-IgG binding, and cytotoxicity assay, we have disclosed the key role of aspartate 69 (Asp(69)) of TM2 for NMO-IgG epitope assembly. Mutation of Asp(69) to histidine severely impairs NMO-IgG binding for 85.7% of the NMO patient sera analyzed here. Although Blue Native-PAGE, total internal reflection fluorescence microscopy, and water transport assays indicate that the OAP Asp(69) mutant is similar in structure and function to the wild type, molecular dynamic simulations have revealed that the D(69)H mutation has the effect of altering the structural rearrangements of extracellular loop A. In conclusion, Asp(69) is crucial for the spatial control of loop A, the particular molecular conformation of which enables the assembly of NMO-IgG epitopes. These findings provide additional clues for new strategies for NMO treatment and a wealth of information to better approach NMO pathogenesis.

A new gating site in human aquaporin-4: Insights from molecular dynamics simulations.

Aquaporin-4 (AQP4) is the predominant water channel in different organs and tissues. An alteration of its physiological functioning is responsible for several disorders of water regulation and, thus, is considered an attractive target with a promising therapeutic and diagnostic potential. Molecular dynamics (MD) simulations performed on the AQP4 tetramer embedded in a bilayer of lipid molecules allowed us to analyze the role of spontaneous fluctuations occurring inside the pore. Following the approach by Hashido et al. [Hashido M, Kidera A, Ikeguchi M (2007) Biophys J 93: 373-385], our analysis on 200ns trajectory discloses three domains inside the pore as key elements for water permeation. Herein, we describe the gating mechanism associated with the well-known selectivity filter on the extracellular side of the pore and the crucial regulation ensured by the NPA motifs (asparagine, proline, alanine). Notably, on the cytoplasmic side, we find a putative gate formed by two residues, namely, a cysteine belonging to the loop D (C178) and a histidine from loop B (H95). We observed that the spontaneous reorientation of the imidazole ring of H95 acts as a molecular switch enabling H-bond interaction with C178. The occurrence of such local interaction seems to be responsible for the narrowing of the pore and thus of a remarkable decrease in water flux rate. Our results are in agreement with recent experimental observations and may represent a promising starting point to pave the way for the discovery of chemical modulators of AQP4 water permeability.

Effects of different self-assembled monolayers on thin-film morphology: a combined DFT/MD simulation protocol.

Organic thin film transistors (OTFTs) are multilayer field-effect transistors that employ an organic conjugated material as semiconductor. Several experimental groups have recently demonstrated that the insertion of an organic self-assembled monolayer (SAM) between the dielectric and the semiconductive layer is responsible for a sensible improvement of the OTFT performances in terms of an increased charge carrier mobility caused by a higher degree of order in the organic semiconductor layer. Here, we describe a combined periodic density functional theory (DFT) and classical molecular dynamics (MD) protocol applied to four different SAMs and a pentacene monolayer deposited onto their surfaces. In particular, we investigate the morphology and the surface of the four SAMs and the translational, orientational, and nematic order of the monolayer through the calculation of several distribution functions and order parameters pointing out the differences among the systems and relating them to known experimental results. Our calculations also suggest that small differences in the SAM molecular design will produce remarkable differences in the SAM surface and monolayer order. In particular, our simulations explain how a SAM with a bulky terminal group results in an irregular and rough surface that determines the deposition of a disordered semiconductive monolayer. On the contrary, SAMs with a small terminal group generate smooth surfaces with uninterrupted periodicity, thus favoring the formation of an ordered pentacene monolayer that increases the mobility of charge carriers and improves the overall performances of the OTFT devices. Our results clearly point out that the in silico procedure presented here might be of help in tuning the design of SAMs in order to improve the quality of OTFT devices.

Morphological and charge transport properties of amorphous and crystalline P3HT and PBTTT: insights from theory.

We explore the relation between the morphological and the charge transport properties of poly(3-hexylthiophene) (P3HT) and poly(2,5-bis(3-alkylthiophen-2-yl)thieno[3,2-b]thiophene) (PBTTT) semiconductor polymers in both amorphous and crystalline phases. Using molecular dynamics to simulate bulk supercells and the Marcus theory to analyze the transport properties we found that amorphous systems display a reduced hole mobility due to the loss of nematic order and π-π stacking leading to a reduction in the electronic coupling between two chains. In the crystal phase, PBTTT displays a larger charge mobility than P3HT due to the interdigitation of the side chains enhancing the stability of the conjugated rings on the backbones. This more stable π-π stacking reduces the energetic disorder with respect to P3HT and increases the electronic coupling. In contrast, in the amorphous phase, PBTTT displays a reduced charge mobility with respect to P3HT due to the absence of side chains attached to the thienothiophenes, which increases their fluctuations and the energetic disorder. In addition, we show that it is possible to calculate the reorganization energy neglecting the side chains of the polymers and thus saving computational time. Within this approximation, we obtained mobility values matching the experimental measurements, thus confirming that the side chains are crucial to shape the morphology of the polymeric systems but are not involved in the charge transport process.

Quality assessment and community detection methods for anonymized mobility data in the Italian Covid context.

We discuss how to assess the reliability of partial, anonymized mobility data and compare two different methods to identify spatial communities based on movements: Greedy Modularity Clustering (GMC) and the novel Critical Variable Selection (CVS). These capture different aspects of mobility: direct population fluxes (GMC) and the probability for individuals to move between two nodes (CVS). As a test case, we consider movements of Italians before and during the SARS-Cov2 pandemic, using Facebook users' data and publicly available information from the Italian National Institute of Statistics (Istat) to construct daily mobility networks at the interprovincial level. Using the Perron-Frobenius (PF) theorem, we show how the mean stochastic network has a stationary population density state comparable with data from Istat, and how this ceases to be the case if even a moderate amount of pruning is applied to the network. We then identify the first two national lockdowns through temporal clustering of the mobility networks, define two representative graphs for the lockdown and non-lockdown conditions and perform optimal spatial community identification on both graphs using the GMC and CVS approaches. Despite the fundamental differences in the methods, the variation of information (VI) between them assesses that they return similar partitions of the Italian provincial networks in both situations. The information provided can be used to inform policy, for example, to define an optimal scale for lockdown measures. Our approach is general and can be applied to other countries or geographical scales.

A conformational rearrangement of the SARS-CoV-2 host protein sigma-1 is required for antiviral activity: insights from a combined in-silico/in-vitro approach.

The development of effective drugs to treat coronavirus infections remains a significant challenge for the scientific community. Recent evidence reports on the sigma-1 receptor (S1R) as a key druggable host protein in the SARS-CoV-1 and SARS-CoV-2 interactomes and shows a potent antiviral activity against SARS-CoV-2 for the S1R antagonist PB28. To improve PB28 activity, we designed and tested a series of its analogues and identified a compound that is fourfold more potent against SARS-CoV-2 than PB28 itself. Interestingly, we found no direct correlation between S1R affinity and SARS-CoV-2 antiviral activity. Building on this, we employed comparative induced fit docking and molecular dynamics simulations to gain insights into the possible mechanism that occurs when specific ligand-protein interactions take place and that may be responsible for the observed antiviral activity. Our findings offer a possible explanation for the experimental observations, provide insights into the S1R conformational changes upon ligand binding and lay the foundation for the rational design of new S1R ligands with potent antiviral activity against SARS-CoV-2 and likely other viruses.

Membrane binding of pore-forming γ-hemolysin components studied at different lipid compositions.

Methicillin-resistant Staphylococcus aureus is among those pathogens currently posing the highest threat to public health. Its host immune evasion strategy is mediated by pore-forming toxins (PFTs), among which the bi-component γ-hemolysin is one of the most common. The complexity of the porogenesis mechanism by γ-hemolysin poses difficulties in the development of antivirulence therapies targeting PFTs from S. aureus, and sparse and apparently contrasting experimental data have been produced. Here, through a large set of molecular dynamics simulations at different levels of resolution, we investigate the first step of pore formation, and in particular the effect of membrane composition on the ability of γ-hemolysin components, LukF and Hlg2, to steadily adhere to the lipid bilayer in the absence of proteinaceous receptors. Our simulations are in agreement with experimental data of γ-hemolysin pore formation on model membranes, which are here explained on the basis of the bilayer properties. Our computational investigation suggests a possible rationale to explain experimental data on phospholipid binding to the LukF component, and to hypothesise a mechanism by which, on purely lipidic bilayers, the stable anchoring of LukF to the cell surface facilitates Hlg2 binding, through the exposure of its N-terminal region. We expect that further insights on the mechanism of transition between soluble and membrane bound-forms and on the role played by the lipid molecules will contribute to the design of antivirulence agents with enhanced efficacy against methicillin-resistant S. aureus infections.

Mechanism of action of cyclophilin a explored by metadynamics simulations.

Trans/cis prolyl isomerisation is involved in several biological processes, including the development of numerous diseases. In the HIV-1 capsid protein (CA), such a process takes place in the uncoating and recruitment of the virion and is catalyzed by cyclophilin A (CypA). Here, we use metadynamics simulations to investigate the isomerization of CA's model substrate HAGPIA in water and in its target protein CypA. Our results allow us to propose a novel mechanistic hypothesis, which is finally consistent with all of the available molecular biology data.

G protein inactive and active forms investigated by simulation methods.

Molecular dynamics and computational alanine scanning techniques have been used to investigate G proteins in their inactive state (the Galpha(i1)beta(1)gamma(2) heterotrimer) as well as in their empty and monomeric active states (Galpha(i1) subunit). We find that: (i) the residue Q204 of Galpha(i1) plays a key role for binding Gbeta(1)gamma(2) and is classified among the most relevant in the interaction with a key cellular partner, the so-called regulator of G protein signaling protein. The mutation of this residue to L, which is observed in a variety of diseases, provides still fair stability to the inactive state because of the formation of van der Waals interactions. (ii) The empty state turns out to adopt some structural features of the active one, including a previously unrecognized rearrangement of a key residue (K46). (iii) The so-called Switch IV region increases its mobility on passing from the empty to the active state, and, even more, to the inactive state. Such change in mobility could be important for its several structural and functional roles. (iv) A large scale motion of the helical domain in the inactive state might be important for GDP release upon activation by GPCR, consistently with experimental data.

Three-dimensional microtubule behavior in Xenopus egg extracts reveals four dynamic states and state-dependent elastic properties.

Although microtubules are key players in many cellular processes, very little is known about their dynamic and mechanical properties in physiological three-dimensional environments. The conventional model of microtubule dynamic instability postulates two dynamic microtubule states, growth and shrinkage. However, several studies have indicated that such a model does not provide a comprehensive quantitative and qualitative description of microtubule behavior. Using three-dimensional laser light-sheet fluorescence microscopy and a three-dimensional sample preparation in spacious Teflon cylinders, we measured microtubule dynamic instability and elasticity in interphase Xenopus laevis egg extracts. Our data are inconsistent with a two-state model of microtubule dynamic instability and favor an extended four-state model with two independent metastable pause states over a three-state model with a single pause state. Moreover, our data on kinetic state transitions rule out a simple GTP cap model as the driving force of microtubule stabilization in egg extracts on timescales of a few seconds or longer. We determined the three-dimensional elastic properties of microtubules as a function of both the contour length and the dynamic state. Our results indicate that pausing microtubules are less flexible than growing microtubules and suggest a growth-speed-dependent persistence length. These data might hint toward mechanisms that enable microtubules to efficiently perform multiple different tasks in the cell and suggest the development of a unified model of microtubule dynamics and microtubule mechanics.

Conformations of confined biopolymers.

Nanoscale and microscale confinement of biopolymers naturally occurs in cells and has been recently achieved in artificial structures designed for nanotechnological applications. Here, we present an extensive theoretical investigation of the conformations and shape of a biopolymer with varying stiffness confined to a narrow channel. Combining scaling arguments, analytical calculations, and Monte Carlo simulations, we identify various scaling regimes where master curves quantify the functional dependence of the polymer conformations on the chain stiffness and strength of confinement.