180 research outputs found
Reliable in silico ranking of engineered therapeutic TCR binding affinities with MMPB/GBSA
Accurate and efficient in silico ranking of proteinprotein binding affinities is useful for protein design with applications in biological therapeutics. One popular approach to rank binding affinities is to apply the molecular mechanics Poisson-Boltzmann/generalized Born surface area (MMPB/ GBSA) method to molecular dynamics (MD) trajectories. Here, we identify protocols that enable the reliable evaluation of T-cell receptor (TCR) variants binding to their target, peptide-human leukocyte antigens (pHLAs). We suggest different protocols for variant sets with a few (<= 4) or many mutations, with entropy corrections important for the latter. We demonstrate how potential outliers could be identified in advance and that just 5-10 replicas of short (4 ns) MD simulations may be sufficient for the reproducible and accurate ranking of TCR variants. The protocols developed here can be applied toward in silico screening during the optimization of therapeutic TCRs, potentially reducing both the cost and time taken for biologic development
Lipid Mediators Regulate Pulmonary Fibrosis: Potential Mechanisms and Signaling Pathways
Idiopathic pulmonary fibrosis (IPF) is a progressive lung disease of unknown etiology characterized by distorted distal lung architecture, inflammation, and fibrosis. The molecular mechanisms involved in the pathophysiology of IPF are incompletely defined. Several lung cell types including alveolar epithelial cells, fibroblasts, monocyte-derived macrophages, and endothelial cells have been implicated in the development and progression of fibrosis. Regardless of the cell types involved, changes in gene expression, disrupted glycolysis, and mitochondrial oxidation, dysregulated protein folding, and altered phospholipid and sphingolipid metabolism result in activation of myofibroblast, deposition of extracellular matrix proteins, remodeling of lung architecture and fibrosis. Lipid mediators derived from phospholipids, sphingolipids, and polyunsaturated fatty acids play an important role in the pathogenesis of pulmonary fibrosis and have been described to exhibit pro- and anti-fibrotic effects in IPF and in preclinical animal models of lung fibrosis. This review describes the current understanding of the role and signaling pathways of prostanoids, lysophospholipids, and sphingolipids and their metabolizing enzymes in the development of lung fibrosis. Further, several of the lipid mediators and enzymes involved in their metabolism are therapeutic targets for drug development to treat IPF
Free energy along drug-protein binding pathways interactively sampled in virtual reality
We describe a two-step approach for combining interactive molecular dynamics
in virtual reality (iMD-VR) with free energy (FE) calculation to explore the
dynamics of biological processes at the molecular level. We refer to this
combined approach as iMD-VR-FE. Stage one involves using a state-of-the-art
iMD-VR framework to generate a diverse range of protein-ligand unbinding
pathways, benefitting from the sophistication of human spatial and chemical
intuition. Stage two involves using the iMD-VR-sampled pathways as initial
guesses for defining a path-based reaction coordinate from which we can obtain
a corresponding free energy profile using FE methods. To investigate the
performance of the method, we apply iMD-VR-FE to investigate the unbinding of a
benzamidine ligand from a trypsin protein. The binding free energy calculated
using iMD-VR-FE is similar for each pathway, indicating internal consistency.
Moreover, the resulting free energy profiles can distinguish energetic
differences between pathways corresponding to various protein-ligand
conformations (e.g., helping to identify pathways that are more favourable) and
enable identification of metastable states along the pathways. The two-step
iMD-VR-FE approach offers an intuitive way for researchers to test hypotheses
for candidate pathways in biomolecular systems, quickly obtaining both
qualitative and quantitative insight
On the exact electric and magnetic fields of an electric dipole
We derive from Jefimenko's equations a multipole expansion in order to obtain
the exact expressions for the electric and magnetic fields of an electric
dipole with an arbitrary time dependence. A few comments are also made about
the usual expositions found in most common undergraduate and graduate textbooks
as well as in the literature on this topic
Peptide cargo tunes a network of correlated motions in human leukocyte antigens
Most biomolecular interactions are typically thought to increase the (local) rigidity of a complex, for example, in drugâtarget binding. However, detailed analysis of specific biomolecular complexes can reveal a more subtle interplay between binding and rigidity. Here, we focussed on the human leucocyte antigen (HLA), which plays a crucial role in the adaptive immune system by presenting peptides for recognition by the αÎČ Tâcell receptor (TCR). The role that the peptide plays in tuning HLA flexibility during TCR recognition is potentially crucial in determining the functional outcome of an immune response, with obvious relevance to the growing list of immunotherapies that target the Tâcell compartment. We have applied highâpressure/temperature perturbation experiments, combined with molecular dynamics simulations, to explore the drivers that affect molecular flexibility for a series of different peptideâHLA complexes. We find that different peptide sequences affect peptideâHLA flexibility in different ways, with the peptide cargo tuning a network of correlated motions throughout the pHLA complex, including in areas remote from the peptideâbinding interface, in a manner that could influence Tâcell antigen discrimination
Exposing the Interplay Between Enzyme Turnover, Protein Dynamics and the Membrane Environment in Monoamine Oxidase B
There is an increasing realization that structure-based drug design may show improved success rates by understanding the ensemble of conformations and sub-states accessible to an enzyme and how the environment affects this ensemble. Human monoamine oxidase B (MAO-B) catalyzes the oxidation of amines and is inhibited for the treatment of both Parkinsonâs disease and depression. Despite its clinical importance, its catalytic mechanism remains unclear and routes to drugging this target would be valuable and relevant. Evidence of a radical in either the transition state or resting state of MAO-B is present throughout the literature, and is suggested to be a flavin semiquinone, a tyrosyl radical or both. Here we see evidence of a resting state flavin semiquinone, via absorption redox studies and electron paramagnetic resonance, suggesting that the anionic semiquinone is biologically relevant. Based on enzyme kinetic studies, enzyme variants and molecular dynamics simulations we find evidence for the crucial importance of the membrane environment in mediating the activity of MAO-B and that this mediation is related to effects on the protein dynamics of MAO-B. Further, our MD simulations identify a hitherto undescribed entrance for substrate binding, membrane modulated substrate access, and indications for half-site reactivity: only one active site is accessible to binding at a time. Our study combines both experimental and computational evidence to illustrate the subtle interplay between enzyme activity, protein dynamics and the immediate membrane environment. Understanding key biomedical enzymes to this level of detail will be crucial to inform strategies (and binding sites) for rational drug design for these drug targets
Active site loop engineering abolishes water capture in hydroxylating sesquiterpene synthases
Terpene synthases (TS) catalyze complex reactions to produce a diverse array of terpene skeletons from linear isoprenyl diphosphates. Patchoulol synthase (PTS) from Pogostemon cablin converts farnesyl diphosphate into patchoulol. Using simulation-guided engineering, we obtained PTS variants that eliminate water capture. Further, we demonstrate that modifying the structurally conserved Hα-1 loop also reduces hydroxylation in PTS, as well as in germacradiene-11-ol synthase (Gd11olS), leading to cyclic neutral intermediates as products, including α-bulnesene (PTS) and isolepidozene (Gd11olS). Hα-1 loop modification could be a general strategy for engineering sesquiterpene synthases to produce complex cyclic hydrocarbons without the need for structure determination or modeling
Redesigning the molecular choreography to prevent hydroxylation in Germacradien-11-ol synthase catalysis
Natural sesquiterpene synthases have evolved to make complex terpenoids by quenching reactive carbocations either by proton transfer or by hydroxylation (water capture), depending on their active site. Germacradien-11-ol synthase (Gd11olS) from Streptomyces coelicolor catalyzes the cyclization of farnesyl diphosphate (FDP) into the hydroxylated sesquiterpene germacradien-11-ol. Here, we combine experiment and simulation to guide the redesign of its active site pocket to avoid hydroxylation of the product. Molecular dynamics simulations indicate two regions between which water molecules can flow that are responsible for hydroxylation. Point mutations of selected residues result in variants that predominantly form a complex nonhydroxylated product, which we identify as isolepidozene. Our results indicate how these mutations subtly change the molecular choreography in the Gd11olS active site and thereby pave the way for the engineering of terpene synthases to make complex terpenoid products
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