Most cited article - PubMed ID 28282625
Phosphorylation of the regulatory domain of human tyrosine hydroxylase 1 monitored using non-uniformly sampled NMR
By merging advanced dimensionality reduction (DR) and clustering algorithm (CA) techniques, our study advances the sampling procedure for predicting NMR chemical shifts (CS) in intrinsically disordered proteins (IDPs), making a significant leap forward in the field of protein analysis/modeling. We enhance NMR CS sampling by generating clustered ensembles that accurately reflect the different properties and phenomena encapsulated by the IDP trajectories. This investigation critically assessed different rapid CS predictors, both neural network (e.g., Sparta+ and ShiftX2) and database-driven (ProCS-15), and highlighted the need for more advanced quantum calculations and the subsequent need for more tractable-sized conformational ensembles. Although neural network CS predictors outperformed ProCS-15 for all atoms, all tools showed poor agreement with HN CSs, and the neural network CS predictors were unable to capture the influence of phosphorylated residues, highly relevant for IDPs. This study also addressed the limitations of using direct clustering with collective variables, such as the widespread implementation of the GROMOS algorithm. Clustered ensembles (CEs) produced by this algorithm showed poor performance with chemical shifts compared to sequential ensembles (SEs) of similar size. Instead, we implement a multiscale DR and CA approach and explore the challenges and limitations of applying these algorithms to obtain more robust and tractable CEs. The novel feature of this investigation is the use of solvent-accessible surface area (SASA) as one of the fingerprints for DR alongside previously investigated α carbon distance/angles or ϕ/ψ dihedral angles. The ensembles produced with SASA tSNE DR produced CEs better aligned with the experimental CS of between 0.17 and 0.36 r2 (0.18-0.26 ppm) depending on the system and replicate. Furthermore, this technique produced CEs with better agreement than traditional SEs in 85.7% of all ensemble sizes. This study investigates the quality of ensembles produced based on different input features, comparing latent spaces produced by linear vs nonlinear DR techniques and a novel integrated silhouette score scanning protocol for tSNE DR.
Tauopathies, including Alzheimer's disease (AD), are the most troublesome of all age-related chronic conditions, as there are no well-established disease-modifying therapies for their prevention and treatment. Spatio-temporal distribution of tau protein pathology correlates with cognitive decline and severity of the disease, therefore, tau protein has become an appealing target for therapy. Current knowledge of the pathological effects and significance of specific species in the tau aggregation pathway is incomplete although more and more structural and mechanistic insights are being gained using biophysical techniques. Here, we review the application of NMR to structural studies of various tau forms that appear in its aggregation process, focusing on results obtained from solid-state NMR. Furthermore, we discuss implications from these studies and their prospective contribution to the development of new tauopathy therapies.
- Keywords
- Alzheimer’s disease, filaments, nuclear magnetic resonance, protein structure, tau,
- Publication type
- Journal Article MeSH
- Review MeSH
Biomolecular force fields optimized for globular proteins fail to properly reproduce properties of intrinsically disordered proteins. In particular, parameters of the water model need to be modified to improve applicability of the force fields to both ordered and disordered proteins. Here, we compared performance of force fields recommended for intrinsically disordered proteins in molecular dynamics simulations of three proteins differing in the content of ordered and disordered regions (two proteins consisting of a well-structured domain and of a disordered region with and without a transient helical motif and one disordered protein containing a region of increased helical propensity). The obtained molecular dynamics trajectories were used to predict measurable parameters, including radii of gyration of the proteins and chemical shifts, residual dipolar couplings, paramagnetic relaxation enhancement, and NMR relaxation data of their individual residues. The predicted quantities were compared with experimental data obtained within this study or published previously. The results showed that the NMR relaxation parameters, rarely used for benchmarking, are particularly sensitive to the choice of force-field parameters, especially those defining the water model. Interestingly, the TIP3P water model, leading to an artificial structural collapse, also resulted in unrealistic relaxation properties. The TIP4P-D water model, combined with three biomolecular force-field parameters for the protein part, significantly improved reliability of the simulations. Additional analysis revealed only one particular force field capable of retaining the transient helical motif observed in NMR experiments. The benchmarking protocol used in our study, being more sensitive to imperfections than the commonly used tests, is well suited to evaluate the performance of newly developed force fields.
Microtubule-associated protein 2c (MAP2c) is involved in neuronal development and is less characterized than its homolog Tau, which has various roles in neurodegeneration. Using NMR methods providing single-residue resolution and quantitative comparison, we investigated molecular interactions important for the regulatory roles of MAP2c in microtubule dynamics. We found that MAP2c and Tau significantly differ in the position and kinetics of sites that are phosphorylated by cAMP-dependent protein kinase (PKA), even in highly homologous regions. We determined the binding sites of unphosphorylated and phosphorylated MAP2c responsible for interactions with the regulatory protein 14-3-3ζ. Differences in phosphorylation and in charge distribution between MAP2c and Tau suggested that both MAP2c and Tau respond to the same signal (phosphorylation by PKA) but have different downstream effects, indicating a signaling branch point for controlling microtubule stability. Although the interactions of phosphorylated Tau with 14-3-3ζ are supposed to be a major factor in microtubule destabilization, the binding of 14-3-3ζ to MAP2c enhanced by PKA-mediated phosphorylation is likely to influence microtubule-MAP2c binding much less, in agreement with the results of our tubulin co-sedimentation measurements. The specific location of the major MAP2c phosphorylation site in a region homologous to the muscarinic receptor-binding site of Tau suggests that MAP2c also may regulate processes other than microtubule dynamics.
- Keywords
- 14-3-3 protein, mass spectrometry (MS), microtubule-associated protein (MAP), nuclear magnetic resonance (NMR), protein kinase A (PKA),
- MeSH
- Amino Acid Motifs MeSH
- Phosphorylation MeSH
- Mass Spectrometry MeSH
- Kinetics MeSH
- Rats MeSH
- Magnetic Resonance Spectroscopy MeSH
- Microtubules metabolism MeSH
- Neurons metabolism MeSH
- Cyclic AMP-Dependent Protein Kinases metabolism MeSH
- 14-3-3 Proteins chemistry MeSH
- Microtubule-Associated Proteins chemistry MeSH
- tau Proteins chemistry MeSH
- Signal Transduction MeSH
- Tubulin metabolism MeSH
- Protein Binding MeSH
- Binding Sites MeSH
- Animals MeSH
- Check Tag
- Rats MeSH
- Animals MeSH
- Publication type
- Journal Article MeSH
- Names of Substances
- MAP2 protein, rat MeSH Browser
- Mapt protein, rat MeSH Browser
- Cyclic AMP-Dependent Protein Kinases MeSH
- 14-3-3 Proteins MeSH
- Microtubule-Associated Proteins MeSH
- tau Proteins MeSH
- Tubulin MeSH
