Calcium-Driven In Silico Inactivation of a Human Olfactory Receptor.

Conformational changes as well as molecular determinants related to the activation and inactivation of olfactory receptors are still poorly understood due to the intrinsic difficulties in the structural determination of this GPCR family. Here, we perform, for the first time, the in silico inactivation of human olfactory receptor OR51E2, highlighting the possible role of calcium in this receptor state transition. Using molecular dynamics simulations, we show that a divalent ion in the ion binding site, coordinated by two acidic residues at positions 2.50 and 3.39 conserved across most ORs, stabilizes the receptor in its inactive state. In contrast, protonation of the same two acidic residues is not sufficient to drive inactivation within the microsecond timescale of our simulations. Our findings suggest a novel molecular mechanism for OR inactivation, potentially guiding experimental validation and offering insights into the possible broader role of divalent ions in GPCR signaling.

Proton Affinity and Conformational Integrity of a 24-Atom Triazine Macrocycle across Physiologically Relevant pH.

For 24-atom triazine macrocycles, protonation of the heterocycle leads to a rigid, folded structure presenting a network of hydrogen bonds. These molecules derive from dynamic covalent chemistry wherein triazine monomers bearing a protected hydrazine group and acetal tethered by the amino acid dimerize quantitatively in an acidic solution. Here, lysine is used, and the product is a tetracation. The primary amines of the lysine side chains do not interfere with quantitative yields of the desired bis(hydrazone) at concentrations of 5-125 mg/mL. Mathematical modeling of data derived from titration experiments of the macrocycle reveals that the pKa values of the protonated triazines are 5.6 and 6.7. Changes in chemical shifts of resonances in the 1H NMR spectra corroborate these values and further support assignment of the protonation sites. The pKa values of the lysine side chains are consistent with expectation. Upon deprotonation, the macrocycle enjoys greater conformational freedom as evident from the broadening of resonances in the 1H and 13C NMR spectra indicative of dynamic motion on the NMR time scale and the appearance of additional conformations at room temperature. While well-tempered metadynamics suggests only a modest difference in accessible conformational footprints of the protonated and deprotonated macrocycles, the shift in conformation(s) supports the stabilizing role that the protons adopt in the hydrogen-bonded network.

Multi-eGO: Model Improvements toward the Study of Complex Self-Assembly Processes.

Structure-based models have been instrumental in simulating protein folding and suggesting hypotheses about the mechanisms involved. Nowadays, at least for fast-folding proteins, folding can be simulated in explicit solvent using classical molecular dynamics. However, other self-assembly processes, such as protein aggregation, are still far from being accessible. Recently, we proposed that a hybrid multistate structure-based model, multi-eGO, could help to bridge the gap toward the simulation of out-of-equilibrium, concentration-dependent self-assembly processes. Here, we further improve the model and show how multi-eGO can effectively and accurately learn the conformational ensemble of the amyloid ß42 intrinsically disordered peptide, reproduce the well-established folding mechanism of the B1 immunoglobulin-binding domain of streptococcal protein G, and reproduce the aggregation as a function of the concentration of the transthyretin 105-115 amyloidogenic peptide. We envision that by learning from the dynamics of a few minima, multi-eGO can become a platform for simulating processes inaccessible to other simulation techniques.

Self-assembly of cyclic peptide monolayers by hydrophobic supramolecular hinges.

Supramolecular polymerisation of two-dimensional (2D) materials requires monomers with non-covalent binding motifs that can control the directionality of both dimensions of growth. A tug of war between these propagation forces can bias polymerisation in either direction, ultimately determining the structure and properties of the final 2D ensemble. Deconvolution of the assembly dynamics of 2D supramolecular systems has been widely overlooked, making monomer design largely empirical. It is thus key to define new design principles for suitable monomers that allow the control of the direction and the dynamics of two-dimensional self-assembled architectures. Here, we investigate the sequential assembly mechanism of new monolayer architectures of cyclic peptide nanotubes by computational simulations and synthesised peptide sequences with selected mutations. Rationally designed cyclic peptide scaffolds are shown to undergo hierarchical self-assembly and afford monolayers of supramolecular nanotubes. The particular geometry, the rigidity and the planar conformation of cyclic peptides of alternating chirality allow the orthogonal orientation of hydrophobic domains that define lateral supramolecular contacts, and ultimately direct the propagation of the monolayers of peptide nanotubes. A flexible 'tryptophan hinge' at the hydrophobic interface was found to allow lateral dynamic interactions between cyclic peptides and thus maintain the stability of the tubular monolayer structure. These results unfold the potential of cyclic peptide scaffolds for the rational design of supramolecular polymerisation processes and hierarchical self-assembly across the different dimensions of space.

Lessons Learned from Multiobjective Automatic Optimizations of Classical Three-Site Rigid Water Models Using Microscopic and Macroscopic Target Experimental Observables.

The development of accurate water models is of primary importance for molecular simulations. Despite their intrinsic approximations, three-site rigid water models are still ubiquitously used to simulate a variety of molecular systems. Automatic optimization approaches have been recently used to iteratively refine three-site water models to fit macroscopic (average) thermodynamic properties, providing state-of-the-art three-site models that still present some deviations from the liquid water properties. Here, we show the results obtained by automatically optimizing three-site rigid water models to fit a combination of microscopic and macroscopic experimental observables. We use Swarm-CG, a multiobjective particle-swarm-optimization algorithm, for training the models to reproduce the experimental radial distribution functions of liquid water at various temperatures (rich in microscopic-level information on, e.g., the local orientation and interactions of the water molecules). We systematically analyze the agreement of these models with experimental observables and the effect of adding macroscopic information to the training set. Our results demonstrate how adding microscopic-rich information in the training of water models allows one to achieve state-of-the-art accuracy in an efficient way. Limitations in the approach and in the approximated description of water in these three-site models are also discussed, providing a demonstrative case useful for the optimization of approximated molecular models, in general.

Accurate and Efficient SAXS/SANS Implementation Including Solvation Layer Effects Suitable for Molecular Simulations.

Small-angle X-ray and neutron scattering (SAXS/SANS) provide valuable insights into the structure and dynamics of biomolecules in solution, complementing a wide range of structural techniques, including molecular dynamics simulations. As contrast-based methods, they are sensitive not only to structural properties but also to solvent-solute interactions. Their use in molecular dynamics simulations requires a forward model that should be as fast and accurate as possible. In this work, we demonstrate the feasibility of calculating SAXS and SANS intensities using a coarse-grained representation consisting of one bead per amino acid and three beads per nucleic acid, with form factors that can be corrected on the fly to account for solvation effects at no additional computational cost. By coupling this forward model with molecular dynamics simulations restrained with SAS data, it is possible to determine conformational ensembles or refine the structure and dynamics of proteins and nucleic acids in agreement with the experimental results. To assess the robustness of this approach, we applied it to gelsolin, for which we acquired SAXS data on its closed state, and to a UP1-microRNA complex, for which we used previously collected measurements. Our hybrid-resolution small-angle scattering (hySAS) implementation, being distributed in PLUMED, can be used with atomistic and coarse-grained simulations using diverse restraining strategies.

Automatic Optimization of Lipid Models in the Martini Force Field Using SwarmCG.

After two decades of continued development of the Martini coarse-grained force field (CG FF), further refinment of the already rather accurate Martini lipid models has become a demanding task that could benefit from integrative data-driven methods. Automatic approaches are increasingly used in the development of accurate molecular models, but they typically make use of specifically designed interaction potentials that transfer poorly to molecular systems or conditions different than those used for model calibration. As a proof of concept, here, we employ SwarmCG, an automatic multiobjective optimization approach facilitating the development of lipid force fields, to refine specifically the bonded interaction parameters in building blocks of lipid models within the framework of the general Martini CG FF. As targets of the optimization procedure, we employ both experimental observables (top-down references: area per lipid and bilayer thickness) and all-atom molecular dynamics simulations (bottom-up reference), which respectively inform on the supra-molecular structure of the lipid bilayer systems and on their submolecular dynamics. In our training sets, we simulate at different temperatures in the liquid and gel phases up to 11 homogeneous lamellar bilayers composed of phosphatidylcholine lipids spanning various tail lengths and degrees of (un)saturation. We explore different CG representations of the molecules and evaluate improvements a posteriori using additional simulation temperatures and a portion of the phase diagram of a DOPC/DPPC mixture. Successfully optimizing up to ∼80 model parameters within still limited computational budgets, we show that this protocol allows the obtainment of improved transferable Martini lipid models. In particular, the results of this study demonstrate how a fine-tuning of the representation and parameters of the models may improve their accuracy and how automatic approaches, such as SwarmCG, may be very useful to this end.

Machine Learning-Based Modeling of Olfactory Receptors in Their Inactive State: Human OR51E2 as a Case Study.

Atomistic-level investigation of olfactory receptors (ORs) is a challenging task due to the experimental/computational difficulties in the structural determination/prediction for members of this family of G-protein coupled receptors. Here, we have developed a protocol that performs a series of molecular dynamics simulations from a set of structures predicted de novo by recent machine learning algorithms and apply it to a well-studied receptor, the human OR51E2. Our study demonstrates the need for simulations to refine and validate such models. Furthermore, we demonstrate the need for the sodium ion at a binding site near D2.50 and E3.39 to stabilize the inactive state of the receptor. Considering the conservation of these two acidic residues across human ORs, we surmise this requirement also applies to the other ∼400 members of this family. Given the almost concurrent publication of a CryoEM structure of the same receptor in the active state, we propose this protocol as an in silico complement to the growing field of ORs structure determination.

Metadynamics simulations of ligands binding to protein surfaces: a novel tool for rational drug design.

Structure-based drug design protocols may encounter difficulties to investigate poses when the biomolecular targets do not exhibit typical binding pockets. In this study, by providing two concrete examples from our labs, we suggest that the combination of metadynamics free energy methods (validated against affinity measurements), along with experimental structural information (by X-ray crystallography and NMR), can help to identify the poses of ligands on protein surfaces. The simulation workflow proposed here was implemented in a widely used code, namely GROMACS, and it could straightforwardly be applied to various drug-design campaigns targeting ligands' binding to protein surfaces.

A Model for the Rapid Assessment of Solution Structures for 24-Atom Macrocycles: The Impact of ß-Branched Amino Acids on Conformation.

Experiment and computation are used to develop a model to rapidly predict solution structures of macrocycles sharing the same Murcko framework. These 24-atom triazine macrocycles result from the quantitative dimerization of identical monomers presenting a hydrazine group and an acetal tethered to an amino acid linker. Monomers comprising glycine and the ß-branched amino acids threonine, valine, and isoleucine yield macrocycles G-G, T-T, V-V, and I-I, respectively. Elements common to all members of the framework include the efficiency of macrocyclization (quantitative), the solution- and solid-state structures (folded), the site of protonation (opposite the auxiliary dimethylamine group), the geometry of the hydrazone (E), the C2 symmetry of the subunits (conserved), and the rotamer state adopted. In aggregate, the data reveal metrics predictive of the three-dimensional solution structure that derive from the fingerprint region of the 1D 1H spectrum and a network of rOes from a single resonance. The metrics also afford delineation of more nuanced structural features that allow subpopulations to be identified among the members of the framework. Well-tempered metadynamics provides free energy surfaces and population distributions of these macrocycles. The areas of the free energy surface decrease with increasing steric bulk (G-G > V-V ∼ T-T > I-I). In addition, the surfaces are increasingly isoenergetic with decreasing steric bulk (G-G > V-V ∼ T-T > I-I).

Predictions of the Poses and Affinity of a Ligand over the Entire Surface of a NEET Protein: The Case of Human MitoNEET.

Human NEET proteins contain two [2Fe-2S] iron-sulfur clusters, bound to three Cys residues and one His residue. They exist in two redox states. Recently, these proteins have revealed themselves as attractive drug targets for mitochondrial dysfunction-related diseases, such as type 2 diabetes, Wolfram syndrome 2, and cancers. Unfortunately, the lack of information and mechanistic understanding of ligands binding to the whole functional, cytoplasmatic domain has limited rational drug design approaches. Here, we use an enhanced sampling technique, volume-based metadynamics, recently developed by a team involving some of us, to predict the poses and affinity of the 2-benzamido-4-(1,2,3,4-tetrahydronaphthalen-2-yl)-thiophene-3-carboxylate ligand to the entire surface of the cytoplasmatic domain of the human NEET protein mitoNEET (mNT) in an aqueous solution. The calculations, based on the recently published X-ray structure of the complex, are consistent with the measured affinity. The calculated free energy landscape revealed that the ligand can bind in multiple sites and with poses other than the one found in the X-ray. This difference is likely to be caused by crystal packing effects that allow the ligand to interact with multiple adjacent NEET protein copies. Such extra contacts are of course absent in the solution; therefore, the X-ray pose is only transient in our calculations, where the binding free energy correlates with the number of contacts. We further evaluated how the reduction and protonation of the Fe-bound histidine, as well as temperature, can affect ligand binding. Both such modifications introduce the possibility for the ligand to bind in an area of the protein other than the one observed in the X-ray, with no or little impact on affinity. Overall, our study can provide insights on the molecular recognition mechanisms of ligand binding to mNT in different oxidative conditions, possibly helping rational drug design of NEET ligands.

Computational Epitope Prediction and Design for Antibody Development and Detection.

The design of optimized protein antigens is a fundamental step in the development of new vaccine candidates and in the detection of therapeutic antibodies. A fundamental prerequisite is the identification of antigenic regions that are most prone to interact with antibodies, namely, B-cell epitopes. Here, we describe an efficient structure-based computational method for epitope prediction, called MLCE. In this approach, all that is required is the 3D structure of the antigen of interest. MLCE can be applied to glycosylated proteins, facilitating the identification of immunoreactive versus immune-shielding carbohydrates.

Topological origin of the protein folding transition.

In this paper, a geometrical and thermodynamical analysis of the global properties of the potential energy landscape of a minimalistic model of a polypeptide is presented. The global geometry of the potential energy landscape is supposed to contain relevant information about the properties of a given sequence of amino acids, that is, to discriminate between a random heteropolymer and a protein. By considering the SH3 and PYP protein-sequences and their randomized versions it turns out that, in addition to the standard signatures of the folding transition-discriminating between protein sequences of amino acids and random heteropolymer sequences-also peculiar geometric signatures of the equipotential hypersurfaces in configuration space can discriminate between proteins and random heteropolymers. Interestingly, these geometric signatures are the "shadows" of deeper topological changes that take place in correspondence with the protein folding transition. The protein folding transition takes place in systems with a small number of degrees of freedom (very far from the Avogadro number) and in the absence of a symmetry-breaking phenomenon. Nevertheless, seen from the deepest level of topology changes of equipotential submanifolds of phase space, the protein folding transition fully qualifies as a phase transition.

Density-tunable pathway complexity in a minimalistic self-assembly model.

An open challenge in self-assembly is learning how to design systems that can be conditionally guided towards different target structures depending on externally-controlled conditions. Using a theoretical and numerical approach, here we discuss a minimalistic self-assembly model that can be steered towards different types of ordered constructs at the equilibrium by solely tuning a facile selection parameter, namely the density of building blocks. Metadynamics and Langevin dynamics simulations allow us to explore the behavior of the system in and out of equilibrium conditions. We show that the density-driven tunability is encoded in the pathway complexity of the system, and specifically in the competition between two different main self-assembly routes. A comprehensive set of simulations provides insight into key factors allowing to make one self-assembling pathway prevailing on the other (or vice versa), determining the selection of the final self-assembled products. We formulate and validate a practical criterion for checking whether a specific molecular design is predisposed for such density-driven tunability of the products, thus offering a new, broader perspective to realize and harness this facile extrinsic control of conditional self-assembly.

Well-Tempered Metadynamics Simulations Predict the Structural and Dynamic Properties of a Chiral 24-Atom Macrocycle in Solution.

Inspired by therapeutic potential, the molecular engineering of macrocycles is garnering increased interest. Exercising control with design, however, is challenging due to the dynamic behavior that these molecules must demonstrate in order to be bioactive. Herein, the value of metadynamics simulations is demonstrated: the free-energy surfaces calculated reveal folded and flattened accessible conformations of a 24-atom macrocycle separated by barriers of ∼6 kT under experimentally relevant conditions. Simulations reveal that the dominant conformer is folded-an observation consistent with a solid-state structure determined by X-ray crystallography and a network of rOes established by 1H NMR. Simulations suggest that the macrocycle exists as a rapidly interconverting pair of enantiomeric, folded structures. Experimentally, 1H NMR shows a single species at room temperature. However, at lower temperature, the interconversion rate between these enantiomers becomes markedly slower, resulting in the decoalescence of enantiotopic methylene protons into diastereotopic, distinguishable resonances due to the persistence of conformational chirality. The emergence of conformational chirality provides critical experimental support for the simulations, revealing the dynamic nature of the scaffold-a trait deemed critical for oral bioactivity.

Enhanced-Sampling Simulations for the Estimation of Ligand Binding Kinetics: Current Status and Perspective.

The dissociation rate (k off) associated with ligand unbinding events from proteins is a parameter of fundamental importance in drug design. Here we review recent major advancements in molecular simulation methodologies for the prediction of k off. Next, we discuss the impact of the potential energy function models on the accuracy of calculated k off values. Finally, we provide a perspective from high-performance computing and machine learning which might help improve such predictions.

Ephemeral ice-like local environments in classical rigid models of liquid water.

Despite great efforts over the past 50 years, the simulation of water still presents significant challenges and open questions. At room temperature and pressure, the collective molecular interactions and dynamics of water molecules may form local structural arrangements that are non-trivial to classify. Here, we employ a data-driven approach built on Smooth Overlap of Atomic Position (SOAP) that allows us to compare and classify how widely used classical models represent liquid water. Macroscopically, the obtained results are rationalized based on water thermodynamic observables. Microscopically, we directly observe how transient ice-like ordered environments may dynamically/statistically form in liquid water, even above freezing temperature, by comparing the SOAP spectra for different ice structures with those of the simulated liquid systems. This confirms recent ab initio-based calculations but also reveals how the emergence of ephemeral local ice-like environments in liquid water at room conditions can be captured by classical water models.

Multiple Poses and Thermodynamics of Ligands Targeting Protein Surfaces: The Case of Furosemide Binding to mitoNEET in Aqueous Solution.

Human NEET proteins, such as NAF-1 and mitoNEET, are homodimeric, redox iron-sulfur proteins characterized by triple cysteine and one histidine-coordinated [2Fe-2S] cluster. They exist in an oxidized and reduced state. Abnormal release of the cluster is implicated in a variety of diseases, including cancer and neurodegeneration. The computer-aided and structure-based design of ligands affecting cluster release is of paramount importance from a pharmaceutical perspective. Unfortunately, experimental structural information so far is limited to only one ligand/protein complex. This is the X-ray structure of furosemide bound to oxidized mitoNEET. Here we employ an enhanced sampling approach, Localized Volume-based Metadynamics, developed by some of us, to identify binding poses of furosemide to human mitoNEET protein in solution. The binding modes show a high variability within the same shallow binding pocket on the protein surface identified in the X-ray structure. Among the different binding conformations, one of them is in agreement with the crystal structure's one. This conformation might have been overstabilized in the latter because of the presence of crystal packing interactions, absent in solution. The calculated binding affinity is compatible with experimental data. Our protocol can be used in a straightforward manner in drug design campaigns targeting this pharmaceutically important family of proteins.

Porous covalent organic nanotubes and their assembly in loops and toroids.

Carbon nanotubes, and synthetic organic nanotubes more generally, have in recent decades been widely explored for application in electronic devices, energy storage, catalysis and biosensors. Despite noteworthy progress made in the synthesis of nanotubular architectures with well-defined lengths and diameters, purely covalently bonded organic nanotubes have remained somewhat challenging to prepare. Here we report the synthesis of covalently bonded porous organic nanotubes (CONTs) by Schiff base reaction between a tetratopic amine-functionalized triptycene and a linear dialdehyde. The spatial orientation of the functional groups promotes the growth of the framework in one dimension, and the strong covalent bonds between carbon, nitrogen and oxygen impart the resulting CONTs with high thermal and chemical stability. Upon ultrasonication, the CONTs form intertwined structures that go on to coil and form toroidal superstructures. Computational studies give some insight into the effect of the solvent in this assembly process.