Dynamics of Protein-RNA Interfaces Using All-Atom Molecular Dynamics Simulations.

Facing the current challenges posed by human health diseases requires the understanding of cell machinery at a molecular level. The interplay between proteins and RNA is key for any physiological phenomenon, as well protein-RNA interactions. To understand these interactions, many experimental techniques have been developed, spanning a very wide range of spatial and temporal resolutions. In particular, the knowledge of tridimensional structures of protein-RNA complexes provides structural, mechanical, and dynamical pieces of information essential to understand their functions. To get insights into the dynamics of protein-RNA complexes, we carried out all-atom molecular dynamics simulations in explicit solvent on nine different protein-RNA complexes with different functions and interface size by taking into account the bound and unbound forms. First, we characterized structural changes upon binding and, for the RNA part, the change in the puckering. Second, we extensively analyzed the interfaces, their dynamics and structural properties, and the structural waters involved in the binding, as well as the contacts mediated by them. Based on our analysis, the interfaces rearranged during the simulation time showing alternative and stable residue-residue contacts with respect to the experimental structure.

Molecular Origin of Distinct Hydration Dynamics in Double Helical DNA and RNA Sequences.

Water molecules are essential to determine the structure of nucleic acids and mediate their interactions with other biomolecules. Here, we characterize the hydration dynamics of analogous DNA and RNA double helices with unprecedented resolution and elucidate the molecular origin of their differences: first, the localization of the slowest hydration water molecules─in the minor groove in DNA, next to phosphates in RNA─and second, the markedly distinct hydration dynamics of the two phosphate oxygen atoms OR and OS in RNA. Using our Extended Jump Model for water reorientation, we assess the relative importance of previously proposed factors, including the local topography, water bridges, and the presence of ions. We show that the slow hydration dynamics at RNA OR sites is not due to bridging water molecules but is caused by both the larger excluded volume and the stronger initial H-bond next to OR, due to the different phosphate orientations in A-form double helical RNA.

Internal Normal Mode Analysis Applied to RNA Flexibility and Conformational Changes.

We investigated the capability of internal normal modes to reproduce RNA flexibility and predict observed RNA conformational changes and, notably, those induced by the formation of RNA-protein and RNA-ligand complexes. Here, we extended our iNMA approach developed for proteins to study RNA molecules using a simplified representation of the RNA structure and its potential energy. Three data sets were also created to investigate different aspects. Despite all the approximations, our study shows that iNMA is a suitable method to take into account RNA flexibility and describe its conformational changes opening the route to its applicability in any integrative approach where these properties are crucial.

A dynamical view of protein-protein complexes: Studies by molecular dynamics simulations.

Protein-protein interactions are at the basis of many protein functions, and the knowledge of 3D structures of protein-protein complexes provides structural, mechanical and dynamical pieces of information essential to understand these functions. Protein-protein interfaces can be seen as stable, organized regions where residues from different partners form non-covalent interactions that are responsible for interaction specificity and strength. They are commonly described as a peripheral region, whose role is to protect the core region that concentrates the most contributing interactions, from the solvent. To get insights into the dynamics of protein-protein complexes, we carried out all-atom molecular dynamics simulations in explicit solvent on eight different protein-protein complexes of different functional class and interface size by taking into account the bound and unbound forms. On the one hand, we characterized structural changes upon binding of the proteins, and on the other hand we extensively analyzed the interfaces and the structural waters involved in the binding. Based on our analysis, in 6 cases out of 8, the interfaces rearranged during the simulation time, in stable and long-lived substates with alternative residue-residue contacts. These rearrangements are not restricted to side-chain fluctuations in the periphery but also affect the core interface. Finally, the analysis of the waters at the interface and involved in the binding pointed out the importance to take into account their role in the estimation of the interaction strength.

Modelling the active SARS-CoV-2 helicase complex as a basis for structure-based inhibitor design.

The RNA helicase (non-structural protein 13, NSP13) of SARS-CoV-2 is essential for viral replication, and it is highly conserved among the coronaviridae family, thus a prominent drug target to treat COVID-19. We present here structural models and dynamics of the helicase in complex with its native substrates based on thorough analysis of homologous sequences and existing experimental structures. We performed and analysed microseconds of molecular dynamics (MD) simulations, and our model provides valuable insights to the binding of the ATP and ssRNA at the atomic level. We identify the principal motions characterising the enzyme and highlight the effect of the natural substrates on this dynamics. Furthermore, allosteric binding sites are suggested by our pocket analysis. Our obtained structural and dynamical insights are important for subsequent studies of the catalytic function and for the development of specific inhibitors at our characterised binding pockets for this promising COVID-19 drug target.

Progress toward SHAPE Constrained Computational Prediction of Tertiary Interactions in RNA Structure.

As more sequencing data accumulate and novel puzzling genetic regulations are discovered, the need for accurate automated modeling of RNA structure increases. RNA structure modeling from chemical probing experiments has made tremendous progress, however accurately predicting large RNA structures is still challenging for several reasons: RNA are inherently flexible and often adopt many energetically similar structures, which are not reliably distinguished by the available, incomplete thermodynamic model. Moreover, computationally, the problem is aggravated by the relevance of pseudoknots and non-canonical base pairs, which are hardly predicted efficiently. To identify nucleotides involved in pseudoknots and non-canonical interactions, we scrutinized the SHAPE reactivity of each nucleotide of the 188 nt long lariat-capping ribozyme under multiple conditions. Reactivities analyzed in the light of the X-ray structure were shown to report accurately the nucleotide status. Those that seemed paradoxical were rationalized by the nucleotide behavior along molecular dynamic simulations. We show that valuable information on intricate interactions can be deduced from probing with different reagents, and in the presence or absence of Mg2+. Furthermore, probing at increasing temperature was remarkably efficient at pointing to non-canonical interactions and pseudoknot pairings. The possibilities of following such strategies to inform structure modeling software are discussed.

Biasing RNA Coarse-Grained Folding Simulations with Small-Angle X-ray Scattering Data.

RNA molecules can easily adopt alternative structures in response to different environmental conditions. As a result, a molecule's energy landscape is rough and can exhibit a multitude of deep basins. In the absence of a high-resolution structure, small-angle X-ray scattering data (SAXS) can narrow down the conformational space available to the molecule and be used in conjunction with physical modeling to obtain high-resolution putative structures to be further tested by experiments. Because of the low resolution of these data, it is natural to implement the integration of SAXS data into simulations using a coarse-grained representation of the molecule, allowing for much wider searches and faster evaluation of SAXS theoretical intensity curves than with atomistic models. We present here the theoretical framework and the implementation of a simulation approach based on our coarse-grained model HiRE-RNA combined with SAXS evaluations "on-the-fly" leading the simulation toward conformations agreeing with the scattering data, starting from partially folded structures as the ones that can easily be obtained from secondary structure prediction-based tools. We show on three benchmark systems how our approach can successfully achieve high-resolution structures with remarkable similarity with the native structure recovering not only the overall shape, as imposed by SAXS data, but also the details of initially missing base pairs.

Endoglin Is an Endothelial Housekeeper against Inflammation: Insight in ECFC-Related Permeability through LIMK/Cofilin Pathway.

Endoglin (Eng) is an endothelial cell (EC) transmembrane glycoprotein involved in adhesion and angiogenesis. Eng mutations result in vessel abnormalities as observed in hereditary hemorrhagic telangiectasia of type 1. The role of Eng was investigated in endothelial functions and permeability under inflammatory conditions, focusing on the actin dynamic signaling pathway. Endothelial Colony-Forming Cells (ECFC) from human cord blood and mouse lung/aortic EC (MLEC, MAEC) from Eng+/+ and Eng+/- mice were used. ECFC silenced for Eng with Eng-siRNA and ctr-siRNA were used to test tubulogenesis and permeability +/- TNFα and +/- LIM kinase inhibitors (LIMKi). In silico modeling of TNFα-Eng interactions was carried out from PDB IDs 5HZW and 5HZV. Calcium ions (Ca2+) flux was studied by Oregon Green 488 in epifluorescence microscopy. Levels of cofilin phosphorylation and tubulin post-translational modifications were evaluated by Western blot. F-actin and actin-tubulin distribution/co-localization were evaluated in cells by confocal microscopy. Eng silencing in ECFCs resulted in a decrease of cell sprouting by 50 ± 15% (p < 0.05) and an increase in pseudo-tube width (41 ± 4.5%; p < 0.001) compared to control. Upon TNFα stimulation, ECFC Eng-siRNA displayed a significant higher permeability compared to ctr-siRNA (p < 0.01), which is associated to a higher Ca2+ mobilization (p < 0.01). Computational analysis suggested that Eng mitigated TNFα activity. F-actin polymerization was significantly increased in ECFC Eng-siRNA, MAEC+/-, and MLEC+/- compared to controls (p < 0.001, p < 0.01, and p < 0.01, respectively) as well as actin/tubulin distribution (p < 0.01). Furthermore, the inactive form of cofilin (P-cofilin at Ser3) was significantly decreased by 36.7 ± 4.8% in ECFC Eng-siRNA compared to ctr-siRNA (p < 0.001). Interestingly, LIMKi reproduced the absence of Eng on TNFα-induced ECFC-increased permeability. Our data suggest that Eng plays a critical role in the homeostasis regulation of endothelial cells under inflammatory conditions (TNFα), and loss of Eng influences ECFC-related permeability through the LIMK/cofilin/actin rearrangement-signaling pathway.

Induced forms of α2-macroglobulin neutralize heparin and direct oral anticoagulant effects.

Alpha2-macroglobulin (α2M) is a physiological macromolecule that facilitates the clearance of many proteinases, cytokines and growth factors in human. Here, we explored the effect of induced forms of α2M on anticoagulant drugs. Gla-domainless factor Xa (GDFXa) and methylamine (MA)-induced α2M were prepared and characterized by electrophoresis, immunonephelometry, chromogenic, clot waveform and rotational thromboelastometry assays. Samples from healthy volunteers and anticoagulated patients were included. In vivo neutralization of anticoagulants was evaluated in C57Bl/6JRj mouse bleeding-model. Anticoagulant binding sites on induced α2M were depicted by computer-aided energy minimization modeling. GDFXa-induced α2M neutralized dabigatran and heparins in plasma and whole blood. In mice, a single IV dose of GDFXa-induced α2M following anticoagulant administration significantly reduced blood loss and bleeding time. Being far easier to prepare, we investigated the efficacy of MA-induced α2M. It neutralized rivaroxaban, apixaban, dabigatran and heparins in spiked samples in a concentration-dependent manner and in samples from treated patients. Molecular docking analysis evidenced the ability of MA-induced α2M to bind non-covalently these compounds via some deeply buried binding sites. Induced forms of α2M have the potential to neutralize direct oral anticoagulants and heparins, and might be developed as a universal antidote in case of major bleeding or urgent surgery.

A Bit Stickier, a Bit Slower, a Lot Stiffer: Specific vs. Nonspecific Binding of Gal4 to DNA.

Transcription factors regulate gene activity by binding specific regions of genomic DNA thanks to a subtle interplay of specific and nonspecific interactions that is challenging to quantify. Here, we exploit Reflective Phantom Interface (RPI), a label-free biosensor based on optical reflectivity, to investigate the binding of the N-terminal domain of Gal4, a well-known gene regulator, to double-stranded DNA fragments containing or not its consensus sequence. The analysis of RPI-binding curves provides interaction strength and kinetics and their dependence on temperature and ionic strength. We found that the binding of Gal4 to its cognate site is stronger, as expected, but also markedly slower. We performed a combined analysis of specific and nonspecific binding-equilibrium and kinetics-by means of a simple model based on nested potential wells and found that the free energy gap between specific and nonspecific binding is of the order of one kcal/mol only. We investigated the origin of such a small value by performing all-atom molecular dynamics simulations of Gal4-DNA interactions. We found a strong enthalpy-entropy compensation, by which the binding of Gal4 to its cognate sequence entails a DNA bending and a striking conformational freezing, which could be instrumental in the biological function of Gal4.

Using normal mode analysis on protein structural models. How far can we go on our predictions?

Normal mode analysis (NMA) is a fast and inexpensive approach that is largely used to gain insight into functional protein motions, and more recently to create conformations for further computational studies. However, when the protein structure is unknown, the use of computational models is necessary. Here, we analyze the capacity of NMA in internal coordinate space to predict protein motion, its intrinsic flexibility, and atomic displacements, using protein models instead of native structures, and the possibility to use it for model refinement. Our results show that NMA is quite insensitive to modeling errors, but that calculations are strictly reliable only for very accurate models. Our study also suggests that internal NMA is a more suitable tool for the improvement of structural models, and for integrating them with experimental data or in other computational techniques, such as protein docking or more refined molecular dynamics simulations.

Antibiotic export by MexB multidrug efflux transporter is allosterically controlled by a MexA-OprM chaperone-like complex.

The tripartite multidrug efflux system MexAB-OprM is a major actor in Pseudomonas aeruginosa antibiotic resistance by exporting a large variety of antimicrobial compounds. Crystal structures of MexB and of its Escherichia coli homolog AcrB had revealed asymmetric trimers depicting a directional drug pathway by a conformational interconversion (from Loose and Tight binding pockets to Open gate (LTO) for drug exit). It remains unclear how MexB acquires its LTO form. Here by performing functional and cryo-EM structural investigations of MexB at various stages of the assembly process, we unveil that MexB inserted in lipid membrane is not set for active transport because it displays an inactive LTC form with a Closed exit gate. In the tripartite complex, OprM and MexA form a corset-like platform that converts MexB into the active form. Our findings shed new light on the resistance nodulation cell division (RND) cognate partners which act as allosteric factors eliciting the functional drug extrusion.

Allosteric Inhibition of Adenylyl Cyclase Type 5 by G-Protein: A Molecular Dynamics Study.

Adenylyl cyclases (ACs) have a crucial role in many signal transduction pathways, in particular in the intricate control of cyclic AMP (cAMP) generation from adenosine triphosphate (ATP). Using homology models developed from existing structural data and docking experiments, we have carried out all-atom, microsecond-scale molecular dynamics simulations on the AC5 isoform of adenylyl cyclase bound to the inhibitory G-protein subunit Gαi in the presence and in the absence of ATP. The results show that Gαi has significant effects on the structure and flexibility of adenylyl cyclase, as observed earlier for the binding of ATP and Gsα. New data on Gαi bound to the C1 domain of AC5 help explain how Gαi inhibits enzyme activity and obtain insight on its regulation. Simulations also suggest a crucial role of ATP in the regulation of the stimulation and inhibition of AC5.

Biomimetic Nanoarchitectures for Light Harvesting: Self-Assembly of Pyropheophorbide-Peptide Conjugates.

The biological light-harvesting process offers an unlimited source of inspiration. The high level of control, adaptation capability, and efficiency challenge humankind to create artificial biomimicking nanoarchitectures with the same performances to respond to our energy needs. Here, in the extensive search for design principles at the base of efficient artificial light harvesters, an approach based on self-assembly of pigment-peptide conjugates is proposed. The solvent-driven and controlled aggregation of the peptide moieties promotes the formation of a dense network of interacting pigments, giving rise to an excitonic network characterized by intense and spectrally wide absorption bands. The ultrafast dynamics of the nanosystems studied through two-dimensional electronic spectroscopy reveals that the excitation energy is funneled in an ultrafast time range (hundreds of femtoseconds) to a manifold of long-living dark states, thus suggesting the considerable potentiality of the systems as efficient harvesters.

The interplay between molecular flexibility and RNA chemical probing reactivities analyzed at the nucleotide level via an extensive molecular dynamics study.

Determination of the tridimensional structure of ribonucleic acid molecules is fundamental for understanding their function in the cell. A common method to investigate RNA structures of large molecules is the use of chemical probes such as SHAPE (2'-hydroxyl acylation analyzed by primer extension) reagents, DMS (dimethyl sulfate) and CMCT (1-cyclohexyl-3-(2-morpholinoethyl) carbodiimide metho-p-toluene sulfate), the reaction of which is dependent on the local structural properties of each nucleotide. In order to understand the interplay between local flexibility, sugar pucker, canonical pairing and chemical reactivity of the probes, we performed all-atom molecular dynamics simulations on a set of RNA molecules for which both tridimensional structure and chemical probing data are available and we analyzed the correlations between geometrical parameters and the chemical reactivity. Our study confirms that SHAPE reactivity is guided by the local flexibility of the different chemical moieties but suggests that a combination of multiple parameters is needed to better understand the implications of the reactivity at the molecular level. This is also the case for DMS and CMCT for which the reactivity appears to be more complex than commonly accepted.

Internal Coordinate Normal Mode Analysis: A Strategy To Predict Protein Conformational Transitions.

We analyze the capacity of normal mode analysis in internal coordinates' space to generate large-amplitude structural deformations that can describe the conformational changes occurring upon the binding of proteins to other species. We also analyze how many modes need to be studied to capture a given transition and whether a combination of two modes is better than using a single mode. The technique is tested on known unbound-to-bound structural transitions for a set of single- or multidomain proteins. The results suggest that this approach is a promising way to generate structures for protein docking or for more refined molecular dynamics simulations.

A molecular dynamics study of adenylyl cyclase: The impact of ATP and G-protein binding.

Adenylyl cyclases (ACs) catalyze the biosynthesis of cyclic adenosine monophosphate (cAMP) from adenosine triphosphate (ATP) and play an important role in many signal transduction pathways. The enzymatic activity of ACs is carefully controlled by a variety of molecules, including G-protein subunits that can both stimulate and inhibit cAMP production. Using homology models developed from existing structural data, we have carried out all-atom, microsecond-scale molecular dynamics simulations on the AC5 isoform of adenylyl cyclase and on its complexes with ATP and with the stimulatory G-protein subunit Gsα. The results show that both ATP and Gsα binding have significant effects on the structure and flexibility of adenylyl cyclase. New data on ATP bound to AC5 in the absence of Gsα notably help to explain how Gsα binding enhances enzyme activity and could aid product release. Simulations also suggest a possible coupling between ATP binding and interactions with the inhibitory G-protein subunit Gαi.

Linker dependent chirality of solvent induced self-assembled structures of porphyrin-α-helical peptide conjugates.

The solvent-promoted aggregation of porphyrins covalently linked to medium length peptides occurs with the formation of chiral supramolecular structures if the peptide chain can adopt an α-helical secondary structure. The circular dichroism spectra of different porphyrin-peptide conjugates show that the chiral arrangement of the porphyrins in the aggregates does not depend on the screw-sense of the peptide helix. Experimental evidence and molecular dynamic simulations suggest that the linker between the porphyrin and the peptide helix is responsible for the overall chirality of supramolecular structures. In particular when the linker is a chiral α-amino acid it is possible to tune the morphology of the chiral aggregates by inverting the configuration of the chiral center.

Density functional theory of nematic elasticity: softening from the polar order.

Recent experiments have evidenced some unconventional features in the elasticity of nematics, which cannot be explained by standard microscopic theories. Here, in the framework of a second-virial density functional theory, we have developed a general approach, relaxing the usual assumption that the angular distribution of particles with respect to their local director is unaffected by the deformation. We show that, for particles with polar symmetry, a new contribution to the splay and bend deformation free energy arises, associated with the onset of polar order. Calculations for conical and bent-shaped particles reveal dramatic softening of the splay and the bend mode, respectively, which eventually may lead to spontaneous deformation.

Hierarchical Propagation of Chirality through Reversible Polymerization: The Cholesteric Phase of DNA Oligomers.

Unveiling the subtle rules that control the buildup of macroscopic chirality starting from chiral molecular elements is a challenge for theory and computations. In this context, a remarkable phenomenon is the formation of helically twisted nematic (cholesteric) phases, with pitch in the micrometer range, driven by self-assembly of relatively small chiral species into supramolecular semiflexible polymers. We have developed a theoretical framework to connect the cholesteric organization to the shape and chirality of the constituents, described with molecular detail, in this kind of system. The theory has been tested against new accurate measurements for solutions of short DNA duplexes. We show that the cholesteric organization is determined by steric repulsion between duplexes, and we identify distinctive features of linear self-assembly in the temperature and concentration dependence of the pitch.