Crystal structure of archaeal IF5A-DHS complex reveals insights into the hypusination mechanism.

The translation factor IF5A is highly conserved in Eukarya and Archaea and undergoes a unique post-translational hypusine modification by the deoxyhypusine synthase (DHS) enzyme. DHS transfers the butylamine moiety from spermidine to IF5A using NAD as a cofactor, forming a deoxyhypusine intermediate. IF5A is a key player in protein synthesis, preventing ribosome stalling in proline-rich sequences during translation elongation and facilitating translation elongation and termination. Additionally, human eIF5A participates in various essential cellular processes and contributes to cancer metastasis, with inhibiting hypusination showing anti-proliferative effects. The hypusination pathway of IF5A is therefore an attractive new therapeutic target. We elucidated the 2.0 Å X-ray crystal structure of the archaeal DHS-IF5A complex, revealing hetero-octameric architecture and providing a detailed view of the complex active site including the hypusination loop. This structure, along with biophysical data and molecular dynamics simulations, provides new insights into the catalytic mechanism of the hypusination reaction.

Mechanism of RGD-conjugated nanodevice binding to its target protein integrin αVß3 by atomistic molecular dynamics and machine learning.

Active targeting strategies have been proposed to enhance the selective uptake of nanoparticles (NPs) by diseased cells, and recent experimental findings have proven the effectiveness of this approach. However, no mechanistic studies have yet revealed the atomistic details of the interactions between ligand-activated NPs and integrins. As a case study, here we investigate, by means of advanced molecular dynamics simulations (MD) and machine learning methods (namely equilibrium MD, binding free energy calculations and training of self-organized maps), the interaction of a cyclic-RGD-conjugated PEGylated TiO2 NP (the nanodevice) with the extracellular segment of integrin αVß3 (the target), the latter experimentally well-known to be over-expressed in several solid tumors. Firstly, we proved that the cyclic-RGD ligand binding to the integrin pocket is established and kept stable even in the presence of the cumbersome realistic model of the nanodevice. In this respect, the unsupervised machine learning analysis allowed a detailed comparison of the ligand/integrin binding in the presence and in the absence of the nanodevice, which unveiled differences in the chemical features. Then, we discovered that unbound cyclic RGDs conjugated to the NP largely contribute to the interactions between the nanodevice and the integrin. Finally, by increasing the density of cyclic RGDs on the PEGylated TiO2 NP, we observed a proportional enhancement of the nanodevice/target binding. All these findings can be exploited to achieve an improved targeting selectivity and cellular uptake, and thus a more successful clinical outcome.

The AhR Signaling Mechanism: A Structural Point of View.

The Aryl hydrocarbon Receptor (AhR) is a well-known sensor of xenobiotics; moreover, it is considered a promising drug target as it is involved in the regulation of many patho-physiological processes. For these reasons the study of its ligand-activated transcription mechanism has stimulated several studies for over twenty years. In this review we highlight the key role of molecular structural information in understanding the different steps of the signaling mechanism. The architecture of the AhR cytosolic complex, encompassing the hsp90 chaperone protein and the XAP2 and p23 co-chaperones, has become available in the last year thanks to Cryo-EM experiments. The structure of the AhR ligand-binding (PAS-B) domain has remained elusive for a long time; it has been predicted by homology modelling, based on known PAS systems, and its ligand-bound forms were modelled through ligand molecular docking. Although very recently some structural information on this domain has become available, considerable efforts are still needed to determine the binding geometries of the AhR key ligands by experimental high-resolution studies. On the other hand, the dimeric structure of AhR with the ARNT protein, bound to the specific DNA responsive element, was partially determined by X-ray crystallography and it was completed by homology modelling. On the whole the current structural knowledge of the main protein complexes that form over the AhR mechanism opens the way to confirm and further investigate the main steps of the proposed ligand-activated transcription mechanism of the AhR.

Molecular Dynamics for the Optimal Design of Functionalized Nanodevices to Target Folate Receptors on Tumor Cells.

Atomistic details on the mechanism of targeting activity by biomedical nanodevices of specific receptors are still scarce in the literature, where mostly ligand/receptor pairs are modeled. Here, we use atomistic molecular dynamics (MD) simulations, free energy calculations, and machine learning approaches on the case study of spherical TiO2 nanoparticles (NPs) functionalized with folic acid (FA) as the targeting ligand of the folate receptor (FR). We consider different FA densities on the surface and different anchoring approaches, i.e., direct covalent bonding of FA γ-carboxylate or through polyethylene glycol spacers. By molecular docking, we first identify the lowest energy conformation of one FA inside the FR binding pocket from the X-ray crystal structure, which becomes the starting point of classical MD simulations in a realistic physiological environment. We estimate the binding free energy to be compared with the existing experimental data. Then, we increase complexity and go from the isolated FA to a nanosystem decorated with several FAs. Within the simulation time framework, we confirm the stability of the ligand-receptor interaction, even in the presence of the NP (with or without a spacer), and no significant modification of the protein secondary structure is observed. Our study highlights the crucial role played by the spacer, FA protonation state, and density, which are parameters that can be controlled during the nanodevice preparation step.

Structural basis of dimerization of chemokine receptors CCR5 and CXCR4.

G protein-coupled receptors (GPCRs) are prominent drug targets responsible for extracellular-to-intracellular signal transduction. GPCRs can form functional dimers that have been poorly characterized so far. Here, we show the dimerization mechanism of the chemokine receptors CCR5 and CXCR4 by means of an advanced free-energy technique named coarse-grained metadynamics. Our results reproduce binding events between the GPCRs occurring in the minute timescale, revealing a symmetric and an asymmetric dimeric structure for each of the three investigated systems, CCR5/CCR5, CXCR4/CXCR4, and CCR5/CXCR4. The transmembrane helices TM4-TM5 and TM6-TM7 are the preferred binding interfaces for CCR5 and CXCR4, respectively. The identified dimeric states differ in the access to the binding sites of the ligand and G protein, indicating that dimerization may represent a fine allosteric mechanism to regulate receptor activity. Our study offers structural basis for the design of ligands able to modulate the formation of CCR5 and CXCR4 dimers and in turn their activity, with therapeutic potential against HIV, cancer, and immune-inflammatory diseases.

TCDD-Induced Allosteric Perturbation of the AhR:ARNT Binding to DNA.

The aryl hydrocarbon receptor (AhR) is a ligand-activated transcription factor that mediates the biological and toxicological effects of structurally diverse chemicals, including halogenated aromatic hydrocarbons. In this work, we investigate the effects of the binding of the AhR prototypical ligand, TCDD, on the stability of the AhR:ARNT complex, as well as the mechanisms by which ligand-induced perturbations propagate to the DNA recognition site responsible for gene transcription. To this aim, a reliable structural model of the overall quaternary structure of the AhR:ARNT:DRE complex is proposed, based on homology modelling. The model shows very good agreement with a previous one and is supported by experimental evidence. Moreover, molecular dynamics simulations are performed to compare the dynamic behaviour of the AhR:ARNT heterodimer in the presence or absence of the TCDD. Analysis of the simulations, performed by an unsupervised machine learning method, shows that TCDD binding to the AhR PASB domain influences the stability of several inter-domain interactions, in particular at the PASA-PASB interface. The inter-domain communication network suggests a mechanism by which TCDD binding allosterically stabilizes the interactions at the DNA recognition site. These findings may have implications for the comprehension of the different toxic outcomes of AhR ligands and drug design.

Metadynamics simulations for the investigation of drug loading on functionalized inorganic nanoparticles.

Inorganic nanoparticles show promising properties that allow them to be efficiently used as drug carriers. The main limitation in this type of application is currently the drug loading capacity, which can be overcome with a proper functionalization of the nanoparticle surface. In this study, we present, for the first time, a computational approach based on metadynamics to estimate the binding free energy of the doxorubicin drug (DOX) to a functionalized TiO2 nanoparticle under different pH conditions. On a thermodynamic basis, we demonstrate the robustness of our approach to capture the overall mechanism behind the pH-triggered release of DOX due to environmental pH changes. Notably, binding free energy estimations align well with what is expected for a pH-sensitive drug delivery system. Based on our results, we envision the use of metadynamics as a promising computational tool for the rational design and in silico optimization of organic ligands with improved drug carrier properties.

SARS-CoV-2 multi-variant rapid detector based on graphene transistor functionalized with an engineered dimeric ACE2 receptor.

Reliable point-of-care (POC) rapid tests are crucial to detect infection and contain the spread of Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2). The emergence of several variants of concern (VOC) can reduce binding affinity to diagnostic antibodies, limiting the efficacy of the currently adopted tests, while showing unaltered or increased affinity for the host receptor, angiotensin converting enzyme 2 (ACE2). We present a graphene field-effect transistor (gFET) biosensor design, which exploits the Spike-ACE2 interaction, the crucial step for SARS-CoV-2 infection. Extensive computational analyses show that a chimeric ACE2-Fragment crystallizable (ACE2-Fc) construct mimics the native receptor dimeric conformation. ACE2-Fc functionalized gFET allows in vitro detection of the trimeric Spike protein, outperforming functionalization with a diagnostic antibody or with the soluble ACE2 portion, resulting in a sensitivity of 20 pg/mL. Our miniaturized POC biosensor successfully detects B.1.610 (pre-VOC), Alpha, Beta, Gamma, Delta, Omicron (i.e., BA.1, BA.2, BA.4, BA.5, BA.2.75 and BQ.1) variants in isolated viruses and patient's clinical nasopharyngeal swabs. The biosensor reached a Limit Of Detection (LOD) of 65 cps/mL in swab specimens of Omicron BA.5. Our approach paves the way for a new and reusable class of highly sensitive, rapid and variant-robust SARS-CoV-2 detection systems.

Insight into the Initial Stages of the Folding Process in Onconase Revealed by UNRES.

The unfolded state of proteins presents many challenges to elucidate the structural basis for biological function. This state is characterized by a large degree of structural heterogeneity which makes it difficult to generate structural models. However, recent experiments into the initial folding events of the 104-residue ribonuclease homologue onconase (ONC) were able to identify the regions in the protein that participate in the initial folding of this protein. Therefore, to gain additional structural insight into the unfolded state of proteins, this study utilized molecular dynamics simulations using the UNited-RESidue (UNRES) force field to evaluate whether there is a good agreement between the experimentally determined initial structures and the structures identified by computer simulations along a folding pathway. Indeed, these UNRES simulations accurately identified the two regions experimentally observed to form the initial native structure along the folding pathway of ONC. In addition, these regions are determined to be chain folding initiation sites (CFIS) according to methods developed previously. Subsequent self-organization maps (SOM) analysis has revealed key structural states involved in these early folding events.

Recognizing the Binding Pattern and Dissociation Pathways of the p300 Taz2-p53 TAD2 Complex.

The dynamic association and dissociation between proteins are the basis of cellular signal transduction. This process becomes much more complicated if one or both interaction partners are intrinsically disordered because intrinsically disordered proteins can undergo disorder-to-order transitions upon binding to their partners. p53, a transcription factor with disordered regions, plays significant roles in many cellular signaling pathways. It is critical to understand the binding/unbinding mechanism involving these disordered regions of p53 at the residue level to reveal how p53 performs its biological functions. Here, we studied the dissociation process of the intrinsically disordered N-terminal transactivation domain 2 (TAD2) of p53 and the transcriptional adaptor zinc-binding 2 (Taz2) domain of transcriptional coactivator p300 using a combination of classical molecular dynamics, steered molecular dynamics, self-organizing maps, and time-resolved force distribution analysis (TRFDA). We observed two different dissociation pathways with different probabilities. One dissociation pathway starts from the TAD2 N-terminus and propagates to the α-helix and finally the C-terminus. The other dissociation pathway is in the opposite order. Subsequent TRFDA results reveal that key residues in TAD2 play critical roles. Besides the residues in agreement with previous experimental results, we also highlighted some other residues that play important roles in the disassociation process. In the dissociation process, non-native interactions were formed to partially compensate for the energy loss due to the breaking of surrounding native interactions. Moreover, our statistical analysis results of other experimentally determined complex structures involving either Taz2 or TAD2 suggest that the binding of the Taz2-TAD2 complex is mainly governed by the binding site of Taz2, which includes three main binding regions. Therefore, the complexes involving Taz2 may follow similar binding/unbinding behaviors, which could be studied together to generate common principles.

The mTOR/4E-BP1/eIF4E Signalling Pathway as a Source of Cancer Drug Targets.

The mechanistic/mammalian target of rapamycin (mTOR) is the crucial hub of signalling pathways that regulate essential steps in the cell life cycle. Once incorporated in the mTORC1 complex, mTOR phosphorylates the eukaryotic initiation factor 4E (eIF4E)- binding protein 1 (4E-BP1), which then releases eIF4E. When not bound to 4EBPs, eIF4E recognizes the mRNA 5'-cap structure and, together with eIF4A and eIF4G, it forms the eIF4F complex that recruits the ribosome on the mRNA. Under normal conditions, the cellular concentration of eIF4E is very low, making eIF4E the limiting factor in the initiation of protein synthesis. The vast majority of cancer types are characterized by the simultaneous deregulation of the mTOR/4E-BP1 signalling pathway and upregulation of eIF4E, which lead to an increased expression of cancer-promoting genes and deregulated cellular growth. Over the last decades, a growing number of selective inhibitors of the mTOR/4E-BP1/eIF4E pathway have been discovered or designed. Several inhibitors with encouraging preclinical results have been tested in clinical trials. This review summarizes the most recent research on drug development against mTOR, 4E-BP1, and eIF4E, describing the design rationale and the available structural and functional data on the most promising compounds.

PathDetect-SOM: A Neural Network Approach for the Identification of Pathways in Ligand Binding Simulations.

Understanding the process of ligand-protein recognition is important to unveil biological mechanisms and to guide drug discovery and design. Enhanced-sampling molecular dynamics is now routinely used to simulate the ligand binding process, resulting in the need for suitable tools for the analysis of large data sets of binding events. Here, we designed, implemented, and tested PathDetect-SOM, a tool based on self-organizing maps to build concise visual models of the ligand binding pathways sampled along single simulations or replicas. The tool performs a geometric clustering of the trajectories and traces the pathways over an easily interpretable 2D map and, using an approximate transition matrix, it can build a graph model of concurrent pathways. The tool was tested on three study cases representing different types of problems and simulation techniques. A clear reconstruction of the sampled pathways was derived in all cases, and useful information on the energetic features of the processes was recovered. The tool is available at https://github.com/MottaStefano/PathDetect-SOM.

Control of the eIF4E activity: structural insights and pharmacological implications.

The central role of eukaryotic translation initiation factor 4E (eIF4E) in controlling mRNA translation has been clearly assessed in the last decades. eIF4E function is essential for numerous physiological processes, such as protein synthesis, cellular growth and differentiation; dysregulation of its activity has been linked to ageing, cancer onset and progression and neurodevelopmental disorders, such as autism spectrum disorder (ASD) and Fragile X Syndrome (FXS). The interaction between eIF4E and the eukaryotic initiation factor 4G (eIF4G) is crucial for the assembly of the translational machinery, the initial step of mRNA translation. A well-characterized group of proteins, named 4E-binding proteins (4E-BPs), inhibits the eIF4E-eIF4G interaction by competing for the same binding site on the eIF4E surface. 4E-BPs and eIF4G share a single canonical motif for the interaction with a conserved hydrophobic patch of eIF4E. However, a second non-canonical and not conserved binding motif was recently detected for eIF4G and several 4E-BPs. Here, we review the structural features of the interaction between eIF4E and its molecular partners eIF4G and 4E-BPs, focusing on the implications of the recent structural and biochemical evidence for the development of new therapeutic strategies. The design of novel eIF4E-targeting molecules that inhibit translation might provide new avenues for the treatment of several conditions.

Exploring the drug loading mechanism of photoactive inorganic nanocarriers through molecular dynamics simulations.

Inorganic nanoparticles are gaining increasing attention as drug carriers because they respond to external physical stimuli, allowing therapy to be combined with diagnosis. Their drawback is low drug loading capacity, which can be improved by proper and efficacious functionalization. In this computational study, we take TiO2 spherical nanoparticles as prototype photoresponsive inorganic nanoparticles and we fully decorate them with two different types of bifunctional ligands: TETTs and DOPACs, which present different surface anchoring groups (silanol or catechol) but the same drug tethering COOH group, although in different concentrations (3 vs. 1), thus causing different steric hindrances. Then, we put these two types of nanocarriers in bulk water and in the presence of several DOX molecules and let the systems evolve through molecular dynamics (MD) simulations, clearly observing drug loading on the nanocarriers. This comparative MD study allows the investigation of the loading mechanism, performance of a conformational analysis and establishment of the guiding interactions through an energy decomposition analysis. We learn that DOX mostly interacts with the functionalized NPs through electrostatics, as a consequence of the protonated amino group, although several H-bonds are also established both with the ligands and with the oxide surface. Different ligands induce a different electrostatic potential around the NP; therefore, those which lead to the formation of more negative hotspots (here TETTs) are found to favour DOX binding. The leading role of electrostatics can provide a rational explanation for a pH-dependent drug release mechanism that is often invoked for DOX when reaching diseased cells because under anomalous acidic conditions both the NP surface and the carboxylate groups of the ligands are expected to get protonated, which of course would weaken, if not totally quench, the interaction of the nanocarrier with protonated DOX.

Metadynamics-Based Approaches for Modeling the Hypoxia-Inducible Factor 2α Ligand Binding Process.

Several methods based on enhanced-sampling molecular dynamics have been proposed for studying ligand binding processes. Here, we developed a protocol that combines the advantages of steered molecular dynamics (SMD) and metadynamics. While SMD is proposed for investigating possible unbinding pathways of the ligand and identifying the preferred one, metadynamics, with the path collective variable (PCV) formalism, is suggested to explore the binding processes along the pathway defined on the basis of SMD, by using only two CVs. We applied our approach to the study of binding of two known ligands to the hypoxia-inducible factor 2α, where the buried binding cavity makes simulation of the process a challenging task. Our approach allowed identification of the preferred entrance pathway for each ligand, highlighted the features of the bound and intermediate states in the free-energy surface, and provided a binding affinity scale in agreement with experimental data. Therefore, it seems to be a suitable tool for elucidating ligand binding processes of similar complex systems.

Reconstruction of ARNT PAS-B Unfolding Pathways by Steered Molecular Dynamics and Artificial Neural Networks.

Several experimental studies indicated that large conformational changes, including partial domain unfolding, have a role in the functional mechanisms of the basic helix loop helix Per/ARNT/SIM (bHLH-PAS) transcription factors. Recently, single-molecule atomic force microscopy (AFM) revealed two distinct pathways for the mechanical unfolding of the ARNT PAS-B. In this work we used steered molecular dynamics simulations to gain new insights into this process at an atomistic level. To reconstruct and classify pathways sampled in multiple simulations, we designed an original approach based on the use of self-organizing maps (SOMs). This led us to identify two types of unfolding pathways for the ARNT PAS-B, which are in good agreement with the AFM findings. Analysis of average forces mapped on the SOM revealed a stable conformation of the PAS-B along one pathway, which represents a possible structural model for the intermediate state detected by AFM. The approach here proposed will facilitate the study of other signal transmission mechanisms involving the folding/unfolding of PAS domains.

Population Dynamics and Structural Effects at Short and Long Range Support the Hypothesis of the Selective Advantage of the G614 SARS-CoV-2 Spike Variant.

SARS-CoV-2 epidemics quickly propagated worldwide, sorting virus genomic variants in newly established propagules of infections. Stochasticity in transmission within and between countries or an actual selective advantage could explain the global high frequency reached by some genomic variants. Using statistical analyses, demographic reconstructions, and molecular dynamics simulations, we show that the globally invasive G614 spike variant 1) underwent a significant demographic expansion in most countries explained neither by stochastic effects nor by overrepresentation in clinical samples, 2) increases the spike S1/S2 furin-like site conformational plasticity (short-range effect), and 3) modifies the internal motion of the receptor-binding domain affecting its cross-connection with other functional domains (long-range effect). Our results support the hypothesis of a selective advantage at the basis of the spread of the G614 variant, which we suggest may be due to structural modification of the spike protein at the S1/S2 proteolytic site, and provide structural information to guide the design of variant-specific drugs.

NURA: A curated dataset of nuclear receptor modulators.

Nuclear receptors (NRs) are key regulators of human health and constitute a relevant target for medicinal chemistry applications as well as for toxicological risk assessment. Several open databases dedicated to small molecules that modulate NRs exist; however, depending on their final aim (i.e., adverse effect assessment or drug design), these databases contain a different amount and type of annotated molecules, along with a different distribution of experimental bioactivity values. Stemming from these considerations, in this work we aim to provide a unified dataset, NURA (NUclear Receptor Activity) dataset, collecting curated information on small molecules that modulate NRs, to be intended for both pharmacological and toxicological applications. NURA contains bioactivity annotations for 15,247 molecules and 11 selected NRs, and it was obtained by integrating and curating data from toxicological and pharmacological databases (i.e., Tox21, ChEMBL, NR-DBIND and BindingDB). Our results show that NURA dataset is a useful tool to bridge the gap between toxicology- and medicinal-chemistry-related databases, as it is enriched in terms of number of molecules, structural diversity and covered atomic scaffolds compared to the single sources. To the best of our knowledge, NURA dataset is the most exhaustive collection of small molecules annotated for their modulation of the chosen nuclear receptors. NURA dataset is intended to support decision-making in pharmacology and toxicology, as well as to contribute to data-driven applications, such as machine learning. The dataset and the data curation pipeline can be downloaded free of charge on Zenodo at the following DOI: https://doi.org/10.5281/zenodo.3991561.

Dopamine-Decorated TiO2 Nanoparticles in Water: A QM/MM vs an MM Description.

Nanoparticle functionalization is a modern strategy in nanotechnology to build up devices for several applications. Modeling fully decorated metal oxide nanoparticles of realistic size (few nanometers) in an aqueous environment is a challenging task. In this work, we present a case study relevant for solar-light exploitation and for biomedical applications, i.e., a dopamine-functionalized TiO2 nanoparticle (1700 atoms) in bulk water, for which we have performed an extensive comparative investigation with both MM and QM/MM approaches of the structural properties and of the conformational dynamics. We have used a combined multiscale protocol for a more efficient exploration of the complex conformational space. On the basis of the results of this study and of some QM and experimental data, we have defined strengths and limitations of the existing force field parameters. Our findings will be useful for an improved modeling and simulation of many other similar hybrid bioinorganic nanosystems in an aqueous environment that are pivotal in a broad range of nanotechnological applications.

Transitional States in Ligand-Dependent Transformation of the Aryl Hydrocarbon Receptor into Its DNA-Binding Form.

The aryl hydrocarbon receptor (AhR) is a ligand-activated transcription factor that mediates the biological and toxicological effects of an AhR lacking the entire PASB structurally diverse chemicals, including halogenated aromatic hydrocarbons. Ligand-dependent transformation of the AhR into its DNA binding form involves a ligand-dependent conformational change, heat shock protein 90 (hsp90), dissociation from the AhR complex and AhR dimerization with the AhR nuclear translocator (ARNT) protein. The mechanism of AhR transformation was examined using mutational approaches and stabilization of the AhR:hsp90 complex with sodium molybdate. Insertion of a single mutation (F281A) in the hsp90-binding region of the AhR resulted in its constitutive (ligand-independent) transformation/DNA binding in vitro. Mutations of AhR residues within the Arg-Cys-rich region (R212A, R217A, R219A) and Asp371 (D371A) impaired AhR transformation without a significant effect on ligand binding. Stabilization of AhR:hsp90 binding with sodium molybdate decreased transformation/DNA binding of the wild type AhR but had no effect on constitutively active AhR mutants. Interestingly, transformation of the AhR in the presence of molybdate allowed detection of an intermediate transformation ternary complex containing hsp90, AhR, and ARNT. These results are consistent with a stepwise transformation mechanism in which binding of ARNT to the liganded AhR:hsp90 complex results in a progressive displacement of hsp90 and conversion of the AhR into its high affinity DNA binding form. The available molecular insights into the signaling mechanism of other Per-ARNT-Sim (PAS) domains and structural information on hsp90 association with other client proteins are consistent with the proposed transformation mechanism of the AhR.