We have developed a suite of protein redesign algorithms that improves realistic in silico modeling of proteins. These algorithms are based on three characteristics that make them unique: (1) ...improved flexibility of the protein backbone, protein side-chains, and ligand to accurately capture the conformational changes that are induced by mutations to the protein sequence; (2) modeling of proteins and ligands as ensembles of low-energy structures to better approximate binding affinity; and (3) a globally optimal protein design search, guaranteeing that the computational predictions are optimal with respect to the input model. Here, we illustrate the importance of these three characteristics. We then describe OSPREY, a protein redesign suite that implements our protein design algorithms. OSPREY has been used prospectively, with experimental validation, in several biomedically relevant settings. We show in detail how OSPREY has been used to predict resistance mutations and explain why improved flexibility, ensembles, and provability are essential for this application.
OSPREY is free and open source under a Lesser GPL license. The latest version is OSPREY 2.0. The program, user manual, and source code are available at www.cs.duke.edu/donaldlab/software.php.
osprey@cs.duke.edu.
Electron Paramagnetic Resonance is a spectroscopic technique which, in combination with site-directed spin-labeling, provides structural and dynamic information about proteins in conditions similar ...to those of their physiological environment. The information is sequence-resolved, as it is based on probing the local dynamics of a paramagnetic label incorporated as a side chain of a selected amino acid. EPR does not impose a limit on the size of the protein or protein complex, as long as it is amenable to site-directed mutagenesis, and is able to obtain reliable distance distributions between two or more labels (identical or different).. The mean value, width and shape of distance distributions, as well as their dependence upon the state of the protein or interactions with physiological partners, provide insight into order-disorder transitions and the roles of protein flexibility.
The main potentialities and limitations of the technique are revised and illustrated with examples of proteins for which order-disorder play an important role.
•Electron Paramagnetic Resonance reports on local protein flexibility and disorder.•Site-Directed Spin-Labeling and EPR are used to assess back-bone dynamics.•DEER obtains distance distributions between spin labels in disordered proteins.•Changes in EPR spectra inform about order-disorder transitions in proteins.•Integrating data and modeling tools provide structural and dynamic models for IDPs.
The molecular basis for the diversity across influenza strains is poorly understood. To gain insight into this question, we mutagenized the viral genome and sequenced recoverable viruses. Only two ...small regions in the genome were enriched for insertions, the hemagglutinin head and the immune-modulatory nonstructural protein 1. These proteins play a major role in host adaptation, and thus need to be able to evolve rapidly. We propose a model in which certain influenza A virus proteins (or protein domains) exist as highly plastic scaffolds, which will readily accept mutations yet retain their functionality. This model implies that the ability to rapidly acquire mutations is an inherent aspect of influenza HA and nonstructural protein 1 proteins; further, this may explain why rapid antigenic drift and a broad host range is observed with influenza A virus and not with some other RNA viruses.
Protein-ligand interactions play key roles in various metabolic pathways, and the proteins involved in these interactions represent major targets for drug discovery. Molecular docking is widely used ...to predict the structure of protein-ligand complexes, and protein flexibility stands out as one of the most important and challenging issues for binding mode prediction. Various docking methods accounting for protein flexibility have been proposed, tackling problems of ever-increasing dimensionality.
This paper presents an overview of conformational sampling methods treating target flexibility during molecular docking. Special attention is given to approaches considering full protein flexibility. Contrary to what is frequently done, this review does not rely on classical biomolecular recognition models to classify existing docking methods. Instead, it applies algorithmic considerations, focusing on the level of flexibility accounted for. This review also discusses the diversity of docking applications, from virtual screening (VS) of small drug-like compounds to geometry prediction (GP) of protein-peptide complexes.
Considering the diversity of docking methods presented here, deciding which one is the best at treating protein flexibility depends on the system under study and the research application. In VS experiments, ensemble docking can be used to implicitly account for large-scale conformational changes, and selective docking can additionally consider local binding-site rearrangements. In other cases, on-the-fly exploration of the whole protein-ligand complex might be needed for accurate GP of the binding mode. Among other things, future methods are expected to provide alternative binding modes, which will better reflect the dynamic nature of protein-ligand interactions.
Protein arginine methyltransferase 5 (PRMT5) is an enzyme that can symmetrically dimethylate arginine residues in histones and nonhistone proteins by using S-adenosyl methionine (SAM) as the methyl ...donating cofactor. We have designed a library of SAM analogues and discovered potent, cell-active, and selective spiro diamines as inhibitors of the enzymatic function of PRMT5. Crystallographic studies confirmed a very interesting binding mode, involving protein flexibility, where both the cofactor pocket and part of substrate binding site are occupied by these inhibitors.
Benchmarks for molecular docking have historically focused on re-docking the cognate ligand of a well-determined protein–ligand complex to measure geometric pose prediction accuracy, and measurement ...of virtual screening performance has been focused on increasingly large and diverse sets of target protein structures, cognate ligands, and various types of decoy sets. Here, pose prediction is reported on the Astex Diverse set of 85 protein ligand complexes, and virtual screening performance is reported on the DUD set of 40 protein targets. In both cases, prepared structures of targets and ligands were provided by symposium organizers. The re-prepared data sets yielded results not significantly different than previous reports of Surflex-Dock on the two benchmarks. Minor changes to protein coordinates resulting from complex pre-optimization had large effects on observed performance, highlighting the limitations of cognate ligand re-docking for pose prediction assessment. Docking protocols developed for cross-docking, which address protein flexibility and produce discrete families of predicted poses, produced substantially better performance for pose prediction. Performance on virtual screening performance was shown to benefit by employing and combining multiple screening methods: docking, 2D molecular similarity, and 3D molecular similarity. In addition, use of multiple protein conformations significantly improved screening enrichment.
To ensure that an external force can break the interaction between a protein and a ligand, the steered molecular dynamics simulation requires a harmonic restrained potential applied to the protein ...backbone. A usual practice is that all or a certain number of protein's heavy atoms or Cα atoms are fixed, being restrained by a small force. This present study reveals that while fixing both either all heavy atoms and or all Cα atoms is not a good approach, while fixing a too small number of few atoms sometimes cannot prevent the protein from rotating under the influence of the bulk water layer, and the pulled molecule may smack into the wall of the active site. We found that restraining the Cα atoms under certain conditions is more relevant. Thus, we would propose an alternative solution in which only the Cα atoms of the protein at a distance larger than 1.2 nm from the ligand are restrained. A more flexible, but not too flexible, protein will be expected to lead to a more natural release of the ligand.
By far the largest proportion of the Earth's biosphere is comprised of organisms that thrive in cold environments (psychrophiles). Their ability to proliferate in the cold is predicated on a capacity ...to synthesize cold-adapted enzymes. These enzymes have evolved a range of structural features that confer a high level of flexibility compared to thermostable homologs. High flexibility, particularly around the active site, is translated into low-activation enthalpy, low-substrate affinity, and high specific activity at low temperatures. High flexibility is also accompanied by a trade-off in stability, resulting in heat lability and, in the few cases studied, cold lability. This review addresses the structure, function, and stability of cold-adapted enzymes, highlighting the challenges for immediate and future consideration. Because of the unique properties of cold-adapted enzymes, they are not only an important focus in extremophile biology, but also represent a valuable model for fundamental research into protein folding and catalysis.
Cytochrome P450 3A4 (CYP3A4) metabolizes a wide range of drugs and toxins. Interactions of CYP3A4 with ligands are difficult to predict due to promiscuity and conformational flexibility. To better ...understand CYP3A4 conformational responses to ligands we use hydrogen deuterium exchange mass spectrometry (HDX-MS) to investigate the effect of ligands on nanodisc-embedded CYP3A4. For a subset of CYP3A4-ligand complexes, differences in the low-frequency modes derived by principal component analyses of molecular dynamics trajectories mirrored the HDX-MS results. The effects of ligands are distributed to flexible elements of CYP3A4 between stretches of secondary structure. The largest effects occur in the F- and G-helices, where most ligands increase the flexibility of the F-helix and connecting loops and decrease the flexibility of the C-term of the G-helix. Most ligands affect the E-F-G, CD and HI regions of the protein. Ligand-dependent differences are observed in the A"-A' loop, BC region, E-helix, K-β1 region, proximal loop, and C-term loop. Correlated HDX responses were observed in the CD region and the C-term of the G-helix that were most pronounced for Type II ligands. Collectively, the HDX and molecular dynamics results suggest that CYP3A4 accommodates diverse binding partners by propagating local backbone fluctuations from the binding site onto the flexible regions of the enzyme via long-range interactions that are differentially modulated by ligands. In contrast to the paradigm wherein ligands decrease protein dynamics at their binding site, a wide range of ligands modestly increase CYP3A4 dynamics throughout the protein including effects remote from the active site.
Superimposition of average structures of CYP3A4 from GaMD simulations of the ligand-free CYP3A4 (tan), CYP3A4-Azimulin complex (blue), and CYP3A4-Ritonavir complex (pink) illustrating the differences in the packing of the F-helix and E-F loop against the E-helix. MD and H/DX mass spectrometry confirm regions uniquely or universally affected by various ligands. Display omitted
•CYP3A4 is a highly promiscuous detoxication enzyme.•H/DX MS reveals CYP3A4 ligands cause globally distributed changes in dynamics.•Results are supported by MD simulations and principle component analysis.•Ligands induce an increase in local dynamics far from the binding site.
Transitions between the unfolded and native states of the ordered globular proteins are accompanied by the accumulation of several intermediates, such as pre-molten globules, wet molten globules, and ...dry molten globules. Structurally equivalent conformations can serve as native functional states of intrinsically disordered proteins. This overview captures the characteristics and importance of these molten globules in both structured and intrinsically disordered proteins. It also discusses examples of engineered molten globules. The formation of these intermediates under conditions of macromolecular crowding and their interactions with nanomaterials are also reviewed.
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