RNA dynamics from experimental and computational approaches.

Conformational dynamics is crucial for the biological function of RNA molecules and for their potential as therapeutic targets. This meeting report outlines key "take-home" messages that emerged from the presentations and discussions during the CECAM workshop "RNA dynamics from experimental and computational approaches" in Paris, June 26-28, 2023.

Assessing RNA atomistic force fields via energy landscape explorations in implicit solvent.

Predicting the structure and dynamics of RNA molecules still proves challenging because of the relative scarcity of experimental RNA structures on which to train models and the very sensitive nature of RNA towards its environment. In the last decade, several atomistic force fields specifically designed for RNA have been proposed and are commonly used for simulations. However, it is not necessarily clear which force field is the most suitable for a given RNA molecule. In this contribution, we propose the use of the computational energy landscape framework to explore the energy landscape of RNA systems as it can bring complementary information to the more standard approaches of enhanced sampling simulations based on molecular dynamics. We apply the EL framework to the study of a small RNA pseudoknot, the Aquifex aeolicus tmRNA pseudoknot PK1, and we compare the results of five different RNA force fields currently available in the AMBER simulation software, in implicit solvent. With this computational approach, we can not only compare the predicted 'native' states for the different force fields, but the method enables us to study metastable states as well. As a result, our comparison not only looks at structural features of low energy folded structures, but provides insight into folding pathways and higher energy excited states, opening to the possibility of assessing the validity of force fields also based on kinetics and experiments providing information on metastable and unfolded states. Supplementary Information: The online version contains supplementary material available at 10.1007/s12551-024-01202-9.

Alpha-2-macroglobulin prevents platelet aggregation induced by brain-derived neurotrophic factor.

The brain-derived neurotrophic factor (BDNF) has been recently shown to have activating effects in isolated platelets. However, BDNF circulates in plasma and a mechanism to preclude constant activation of platelets appears necessary. Hence, we investigated the mechanism regulating BDNF bioavailability in blood. Protein-protein interactions were predicted by molecular docking and validated through immunoprecipitation. Platelet aggregation was assessed using light transmission aggregometry with washed platelets in response to classical agonists or BDNF, in the absence or presence of alpha-2-macroglobulin (α2M), and in platelet-rich plasma. BDNF signaling was assessed with phospho-blots. As little as 25% autologous plasma was sufficient to completely abolish platelet aggregation in response to BDNF. Docking predicted two forms of BDNF binding to native or activated α2M, in parallel and perpendicular arrangements, and the model suggested that the BDNF-α2M complex cannot bind to the high-affinity BDNF receptor, tropomyosin receptor kinase B (TrkB). Experimentally, native and activated α2M formed stable complexes with BDNF preventing BDNF-induced TrkB activation and signal transduction. Both native and activated α2M inhibited BDNF induced-platelet aggregation in a concentration-dependent manner with comparable half-maximal inhibitory concentrations (IC50≈ 125-150 nM). Our study implicates α2M as a physiological regulator of BDNF bioavailability, and as an inhibitor of BDNF-induced platelet activation in blood.

RNA folding pathways from all-atom simulations with a variationally improved history-dependent bias.

Atomically detailed simulations of RNA folding have proven very challenging in view of the difficulties of developing realistic force fields and the intrinsic computational complexity of sampling rare conformational transitions. As a step forward in tackling these issues, we extend to RNA an enhanced path-sampling method previously successfully applied to proteins. In this scheme, the information about the RNA's native structure is harnessed by a soft history-dependent biasing force promoting the generation of productive folding trajectories in an all-atom force field with explicit solvent. A rigorous variational principle is then applied to minimize the effect of the bias. Here, we report on an application of this method to RNA molecules from 20 to 47 nucleotides long and increasing topological complexity. By comparison with analog simulations performed on small proteins with similar size and architecture, we show that the RNA folding landscape is significantly more frustrated, even for relatively small chains with a simple topology. The predicted RNA folding mechanisms are found to be consistent with the available experiments and some of the existing coarse-grained models. Due to its computational performance, this scheme provides a promising platform to efficiently gather atomistic RNA folding trajectories, thus retain the information about the chemical composition of the sequence.

Soluble endoglin reduces thrombus formation and platelet aggregation via interaction with αIIbß3 integrin.

BACKGROUND: The circulating form of human endoglin (sEng) is a cleavage product of membrane-bound endoglin present on endothelial cells. Because sEng encompasses an RGD motif involved in integrin binding, we hypothesized that sEng would be able to bind integrin αIIbß3, thereby compromising platelet binding to fibrinogen and thrombus stability. METHODS: In vitro human platelet aggregation, thrombus retraction, and secretion-competition assays were performed in the presence of sEng. Surface plasmon resonance (SPR) binding and computational (docking) analyses were carried out to evaluate protein-protein interactions. A transgenic mouse overexpressing human sEng (hsEng+) was used to measure bleeding/rebleeding, prothrombin time (PT), blood stream, and embolus formation after FeCl3-induced injury of the carotid artery. RESULTS: Under flow conditions, supplementation of human whole blood with sEng led to a smaller thrombus size. sEng inhibited platelet aggregation and thrombus retraction, interfering with fibrinogen binding, but did not affect platelet activation. SPR binding studies demonstrated that the specific interaction between αIIbß3 and sEng and molecular modeling showed a good fitting between αIIbß3 and sEng structures involving the endoglin RGD motif, suggesting the possible formation of a highly stable αIIbß3/sEng. hsEng+ mice showed increased bleeding time and number of rebleedings compared to wild-type mice. No differences in PT were denoted between genotypes. After FeCl3 injury, the number of released emboli in hsEng+ mice was higher and the occlusion was slower compared to controls. CONCLUSIONS: Our results demonstrate that sEng interferes with thrombus formation and stabilization, likely via its binding to platelet αIIbß3, suggesting its involvement in primary hemostasis control.

Investigating the structural changes due to adenosine methylation of the Kaposi's sarcoma-associated herpes virus ORF50 transcript.

Kaposi's sarcoma-associated herpes virus (KSHV) is a human oncovirus. KSHV relies on manipulating the host cell N6-methyl adenosine (m6A) RNA modification pathway to enhance virus replication. Methylation within a RNA stem loop of the open reading frame 50 (ORF50) increases transcript stability via the recruitment of the m6A reader, SND1. In this contribution we explore the energy landscapes of the unmethylated and methylated RNA stem loops of ORF50 to investigate the effect of methylation on the structure of the stem loop. We observe a significant shift upon methylation between an open and closed configuration of the top of the stem loop. In the unmethylated stem loop the closed configuration is much lower in energy, and, as a result, exhibits higher occupancy.

Computer-aided comprehensive explorations of RNA structural polymorphism through complementary simulation methods.

While RNA folding was originally seen as a simple problem to solve, it has been shown that the promiscuous interactions of the nucleobases result in structural polymorphism, with several competing structures generally observed for non-coding RNA. This inherent complexity limits our understanding of these molecules from experiments alone, and computational methods are commonly used to study RNA. Here, we discuss three advanced sampling schemes, namely Hamiltonian-replica exchange molecular dynamics (MD), ratchet-and-pawl MD and discrete path sampling, as well as the HiRE-RNA coarse-graining scheme, and highlight how these approaches are complementary with reference to recent case studies. While all computational methods have their shortcomings, the plurality of simulation methods leads to a better understanding of experimental findings and can inform and guide experimental work on RNA polymorphism.

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.