1,720,979 research outputs found
Integrating experimental data with molecular simulations to investigate RNA structural dynamics
No full textConformational dynamics is crucial for ribonucleic acid (RNA) function. Techniques such as nuclear magnetic resonance, cryo-electron microscopy, small- and wide-angle X-ray scattering, chemical probing, single-molecule Förster resonance energy transfer, or even thermal or mechanical denaturation experiments probe RNA dynamics at different time and space resolutions. Their combination with accurate atomistic molecular dynamics (MD) simulations paves the way for quantitative and detailed studies of RNA dynamics. First, experiments provide a quantitative validation tool for MD simulations. Second, available data can be used to refine simulated structural ensembles to match experiments. Finally, comparison with experiments allows for improving MD force fields that are transferable to new systems for which data is not available. Here we review the recent literature and provide our perspective on this field
Comparing state-of-the-art approaches to back-calculate SAXS spectra from atomistic molecular dynamics simulations
Full text linkAbstract: Small-angle X-ray scattering (SAXS) experiments are arising as an effective instrument in the structural characterization of biomolecules in solution. However, they suffer from limited resolution, and complementing them with molecular dynamics (MD) simulations can be a successful strategy to obtain information at a finer scale. To this end, tools that allow computing SAXS spectra from MD-sampled structures have been designed over the years, mainly differing in how the solvent contribution is accounted for. In this context, RNA molecules represent a particularly challenging case, as they can have a remarkable effect on the surrounding solvent. Herein, we provide a comparison of SAXS spectra computed through different available software packages for a prototypical RNA system. RNA conformational dynamics is intentionally neglected so as to focus on solvent effects. The results highlight that solvent effects are important also at relatively low scattering vector, suggesting that approaches explicitly modeling solvent contribution are advisable when comparing with experimental data, while more efficient implicit-solvent methods can be a better choice as reaction coordinates to improve MD sampling on-the-fly. Graphic abstract: [Figure not available: see fulltext.
Protein-ligand (un)binding kinetics as a new paradigm for drug discovery at the crossroad between experiments and modelling
No full textIn the last three decades, protein and nucleic acid structure determination and comprehension of the mechanisms, leading to their physiological and pathological functions, have become a cornerstone of biomedical sciences. A deep understanding of the principles governing the fates of cells and tissue at the molecular level has been gained over the years, offering a solid basis for the rational design of drugs aimed at the pharmacological treatment of numerous diseases. Historically, affinity indicators (i.e. Kd and IC50/EC50) have been assumed to be valid indicators of the in vivo efficacy of a drug. However, recent studies pointed out that the kinetics of the drug-receptor binding process could be as important or even more important than affinity in determining the drug efficacy. This eventually led to a growing interest in the characterisation and prediction of the rate constants of protein-ligand association and dissociation. For instance, a drug with a longer residence time can kinetically select a given receptor over another, even if the affinity for both receptors is comparable, thus increasing its therapeutic index. Therefore, understanding the molecular features underlying binding and unbinding processes is of central interest towards the rational control of drug binding kinetics. In this review, we report the theoretical framework behind protein-ligand association and highlight the latest advances in the experimental and computational approaches exploited to investigate the binding kinetics
On the allosteric puzzle and pocket crosstalk through computational means
Full text linkAllostery is a constitutive, albeit often elusive, feature of biomolecular systems, which heavily determines their functioning. Its mechanical, entropic, long-range, ligand, and environment-dependent nature creates far from trivial interplays between residues and, in general, the secondary structure of proteins. This intricate scenario is mirrored in computational terms as different notions of "correlation" among residues and pockets can lead to different conclusions and outcomes. In this article, we put on a common ground and challenge three computational approaches for the correlation estimation task and apply them to three diverse targets of pharmaceutical interest: the androgen A2A receptor, the androgen receptor, and the EGFR kinase domain. Results show that partial results consensus can be attained, yet different notions lead to pointing the attention to different pockets and communications
Integration of computational and experimental biophysics reveals novel insights on the BRCA2 - RAD51 interaction
No full textThe interaction of RAD51 and BRCA2, two proteins involved in the homologous recombination pathway, is a key-process for the integrity of our genome. Through eight short repeats, BRCA2 recruits and transports RAD51 to the sites where DNA damage is processed. Nowadays, only the interaction of RAD51 with BRC4 has been structurally characterized, through X-ray crystallography, by removing the first 97 amino acids of RAD51. Nevertheless, very little biophysical data and no structural information are available on the interaction of the other BRC repeats with RAD51. To shed light on the tight relation between these two proteins we decided to combine experimental and computational approaches. So far, the structural complexity and dynamics of RAD51 have severely hampered our understanding of these interactions. Therefore, we isolated a novel fully human monomeric RAD51 form which was exploited to study the interaction of isolated BRC repeats peptides through orthogonal biophysical experiments. The calculated affinities were then correlated to the ability of the isolated BRC repeats to interact with RAD51 WT, revealing that only peptides with the highest affinities could disassemble RAD51 fibrils. To further rationalize the peptides’ affinities, we performed residue scanning analyses and molecular dynamics (MD) simulations. In particular, the former was applied to estimate the per-residue relative change in binding affinity for each BRC repeat, while MD simulations were used to observe the conformational dynamics associated with the different RAD51-BRC repeat complexes. Interestingly, these results revealed that specific amino-acid variations of the BRC repeats affect their binding to RAD51, and specifically to its N-terminal domain. To support these data and get further insights into the interactions of the full length RAD51 with the BRC repeats, we are aiming to integrate Alphafold2 predictions, SAXS data and MD simulations. As a future perspective we would like to apply this approach, in combination with Cryo-EM, to study the conformational dynamics of BRCA2 truncates containing multiple repeats (e.g. BRC3-4) in complex with RAD51 to unravel novel insights on the role of the spacing regions that separate different BRC repeats
Kinetics of Drug Binding and Residence Time
No full textThe kinetics of drug binding and unbinding is assuming an increasingly crucial role in the long, costly process of bringing a new medicine to patients. For example, the time a drug spends in contact with its biological target is known as residence time (the inverse of the kinetic constant of the drug-target unbinding, 1/kbinfoffeinf). Recent reports suggest that residence time could predict drug efficacy in vivo, perhaps even more effectively than conventional thermodynamic parameters (free energy, enthalpy, entropy). There are many experimental and computational methods for predicting drug-target residence time at an early stage of drug discovery programs. Here, we review and discuss the methodological approaches to estimating drug binding kinetics and residence time. We first introduce the theoretical background of drug binding kinetics from a physicochemical standpoint. We then analyze the recent literature in the field, starting from the experimental methodologies and applications thereof and moving to theoretical and computational approaches to the kinetics of drug binding and unbinding. We acknowledge the central role of molecular dynamics and related methods, which comprise a great number of the computational methods and applications reviewed here. However, we also consider kinetic Monte Carlo. We conclude with the outlook that drug (un)binding kinetics may soon become a go/no go step in the discovery and development of new medicines
Elucidating the BRCA2 - RAD51 interaction by integrating computational and experimental biophysics
No full textThe interaction of RAD51 and BRCA2, two proteins involved in the homologous recombination pathway for the repair of DNA double-strand breaks (DSB), is a key-process for the integrity of our genome [1]. Alterations of their interaction, which have been associated with cancer development, lead to defects in the DNA DSB repair pathway [1]. Through eight short repeats, BRCA2 recruits and transports RAD51 to the sites where DNA damages are processed [2]. Several literature reports highlighted that missense mutations within one BRC repeat can hamper BRCA2 activity [2]. Considering the close homology between the BRC repeats, it is striking how these mutations cannot be counterbalanced by the other non-mutated repeats preserving the function and the interactions of BRCA2 with RAD51. Nowadays, only the interaction of RAD51 with the fourth BRC repeat has been structurally characterized, through X-ray crystallography, by removing the first 97 amino acids of RAD51 [3]. Nevertheless, very little biophysical data and no structural information are available on the interaction of the other BRC repeats with RAD51. Due to the structural complexity and dynamics of RAD51, the mechanistic details of each step of RAD51 recruitment and DNA repair remain elusive. Therefore, the interaction of isolated BRC repeats with RAD51 was thoroughly characterized through orthogonal biophysical experiments exploiting a novel fully human monomeric RAD51, that was isolated in our laboratory. The calculated affinities were then correlated to the ability of the isolated BRC repeats to interact with RAD51 WT, revealing that only peptides with the highest affinities could disassemble RAD51 fibrils. To further rationalize the peptides’ affinities, we performed residue scanning analyses and molecular dynamics (MD) simulations, using the Schrödinger suite of software. These computational approaches were used in a complementary spirit, as they provide static and dynamic information, respectively. In particular, the former was applied to estimate the per-residue relative change in binding affinity for each BRC repeat, while MD simulations were used to observe the conformational dynamics associated with the different RAD51-BRC repeat complexes. Interestingly, these results revealed that specific amino-acid variations of the BRC repeats affect their binding to hRAD51, and specifically to its N-terminal domain. To support these data and get further insights into the interactions of the full length RAD51 with the BRC repeats, SAXS, cross-linking mass spectrometry (XL-MS) experiments, and MD simulations data will be integrated to provide a through characterization of this interaction. Additionally, BRCA2 truncates containing multiple repeats (e.g. BRC3-4) in complex with RAD51 were successfully co-expressed and co-purified. These complexes will be exploited for Cryo-EM and SAXS analysis and will allow us to get further details also on the role of the spacing regions that separate BRC repeats
Combining computational and experimental methods to characterize the RAD51-BRC repeats interaction
No full textGiven its implication in homologous recombination and DNA repair, the interaction between the RAD51 and BRCA2 proteins has gained relevance from a pharmaceutical standpoint. (Davies et al., 2001) However, binding of BRCA2’s BRC repeats increases the flexibility of RAD51’s N-terminal domain, hampering the experimental characterization of the full structure of this complex.(Pellegrini et al., 2002) To overcome this limitation, we combine experimental data from Small Angle X-ray Scattering (SAXS) with Molecular Dynamics (MD) simulations, aiming at reconstructing the conformational ensemble of the RAD51-BRC peptides complexes at atomistic resolution. As initial guess for our simulations, we used an AlphaFold-generated model of the full RAD51 in complex with the BRC4 peptide. The SAXS spectrum computed for this model displayed remarkable disagreement with the experimentally measured one. Therefore, we performed Steered MD simulations to guide the system towards a configuration compatible with the experimental data. Notably, we found that taking into account the solvent contribution(Ballabio et al., 2023) in the calculation of SAXS spectra proved necessary to sample realistic configurations of the complex, and to avoid detachment of the BRC4 peptide from RAD51. We then used enhanced sampling, namely Metadynamics, to generate a heterogeneous ensemble of configurations for subsequent reweighting via the Maximum Entropy(Cesari et al., 2018) principle. Through this strategy, we aim at identifying a conformational ensemble whose average spectrum is compatible with the experimental one
Dissecting the BRCA2 - RAD51 interaction by integrating computational and experimental biophysics
No full textThe interaction of RAD51 and BRCA2, two proteins involved in the homologous recombination pathway for the repair of DNA double-strand breaks (DSB), is a key-process for the integrity of our genome. Alterations of their interaction, which have been associated with cancer development, lead to defects in the DNA DSB repair pathway. Through eight short repeats, BRCA2 recruits and transports RAD51 to the sites where DNA damages are processed. Several literature reports highlighted that missense mutations within one BRC repeat can hamper BRCA2 activity. Considering the close homology between the BRC repeats, it is striking how these mutations cannot be counterbalanced by the other non-mutated repeats preserving the function and the interactions of BRCA2 with RAD51. Nowadays, only the interaction of RAD51 with the fourth BRC repeat has been structurally characterized, through X-ray crystallography, by removing the first 97 amino acids of RAD51. Nevertheless, very little biophysical data and no structural information are available on the interaction of the other BRC repeats with RAD51. My research project aims at elucidating the tight relation between these two proteins by combining experimental and computational approaches. Initially, the interaction of isolated BRC repeats with RAD51 was thoroughly characterized through orthogonal biophysical experiments exploiting a novel fully human monomeric RAD51, that was isolated in our laboratory. The calculated affinities were then correlated to the ability of the isolated BRC repeats to interact with RAD51 WT, revealing that only peptides with the highest affinities could disassemble RAD51 fibrils. To further rationalize the peptides’ affinities, we performed residue scanning analyses and molecular dynamics (MD) simulations, using the Schrödinger suite of software. These computational approaches were used in a complementary spirit, as they provide static and dynamic information, respectively. In particular, the former was applied to estimate the per-residue relative change in binding affinity for each BRC repeat, while MD simulations were used to observe the conformational dynamics associated with the different RAD51-BRC repeat complexes. Interestingly, these results revealed that specific amino-acid variations of the BRC repeats affect their binding to hRAD51, and specifically to its N-terminal domain. To support these data and get further insights into the interactions of the full length RAD51 with the BRC repeats, we are planning to integrate SAXS, cross-linking mass spectrometry (XL-MS) experiments, and MD simulations. Moreover, we were able to co-express and co-purify BRCA2 truncates containing multiple repeats (e.g. BRC3-4) in complex with RAD51. We plan to perform structural investigations on this complex through Cryo-EM, SAXS and computational approaches to further investigate this interaction
Elucidating the RAD51-BRC repeats interaction by integrating computational and experimental approaches
No full textThe interaction between the RAD51 and BRCA2 proteins is central in the homologous recombination pathway and DNA repair; through a series of BRC repeats, BRCA2 recruits RAD51, regulating its polymerization and chaperoning it to the damaged DNA site.1 The binding of the BRC repeats increases the structural flexibility of RAD51’s N-terminal domain, hindering the structural characterization of this complex via experimental methods.2 To reconstruct the conformational ensemble of the RAD51-BRC complex in solution, we combined Molecular Dynamics (MD) simulations with Small Angle X-ray Scattering (SAXS) experimental data. We initially generated an
AlphaFold model of the complex between RAD51 and the BRC4 repeat. The predicted SAXS spectrum for this structure showed significant disagreement with the experimental data. Thus, we used steered MD to guide the structure towards a spectrum compatible with the experimental one. Interestingly, by using a forward model that neglects the solvent contribution for spectra prediction, we were not able to fulfill this task. Specifically, the simulations resulted in the detachment of BRC4 repeat from RAD51, in disagreement with experimental knowledge. Conversely, when including the solvent contribution, the simulations led to a conformation of the RAD51-BRC4 complex in remarkable agreement with the experiments. SAXS experimental spectra are averaged over multiple conformations accessible to the system in solution. Thus, starting from the structure obtained via
steered MD, our next step will be to carry out MD simulations coupled with the maximum entropy principle3 to obtain an ensemble of configurations of the system whose average reflects the experimental SAXS spectrum
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