Unambiguous Tracking of Protein Phosphorylation by Fast High-Resolution FOSY NMR*.

Dysregulation of post-translational modifications (PTMs) like phosphorylation is often involved in disease. NMR may elucidate exact loci and time courses of PTMs at atomic resolution and near-physiological conditions but requires signal assignment to individual atoms. Conventional NMR methods for this base on tedious global signal assignment that may often fail, as for large intrinsically disordered proteins (IDPs). We present a sensitive, robust alternative to rapidly obtain only the local assignment near affected signals, based on FOcused SpectroscopY (FOSY) experiments using selective polarisation transfer (SPT). We prove its efficiency by identifying two phosphorylation sites of glycogen synthase kinase 3 beta (GSK3ß) in human Tau40, an IDP of 441 residues, where the extreme spectral dispersion in FOSY revealed unprimed phosphorylation also of Ser409. FOSY may broadly benefit NMR studies of PTMs and other hotspots in IDPs, including sites involved in molecular interactions.

Prediction of Protein-Protein Binding Interactions in Dimeric Coiled Coils by Information Contained in Folding Energy Landscapes.

Coiled coils represent the simplest form of a complex formed between two interacting protein partners. Their extensive study has led to the development of various methods aimed towards the investigation and design of complex forming interactions. Despite the progress that has been made to predict the binding affinities for protein complexes, and specifically those tailored towards coiled coils, many challenges still remain. In this work, we explore whether the information contained in dimeric coiled coil folding energy landscapes can be used to predict binding interactions. Using the published SYNZIP dataset, we start from the amino acid sequence, to simultaneously fold and dock approximately 1000 coiled coil dimers. Assessment of the folding energy landscapes showed that a model based on the calculated number of clusters for the lowest energy structures displayed a signal that correlates with the experimentally determined protein interactions. Although the revealed correlation is weak, we show that such correlation exists; however, more work remains to establish whether further improvements can be made to the presented model.

Resolution enhancement in NMR spectra by deconvolution with compressed sensing reconstruction.

NMR spectroscopy is one of the basic tools for molecular structure elucidation. Unfortunately, the resolution of the spectra is often limited by inter-nuclear couplings. The existing workarounds often alleviate the problem by trading it for another deficiency, such as spectral artefacts or difficult sample preparation and, thus, are rarely used. We suggest an approach using the coupling deconvolution in the framework of compressed sensing (CS) spectra processing that leads to a major increase in resolution, sensitivity, and overall quality of NUS reconstruction. A new mathematical description of the decoupling by deconvolution explains the effects of thermal noise and reveals a relation with the underlying assumption of the CS. The gain in resolution and sensitivity for challenging molecular systems is demonstrated for the key HNCA experiment used for protein backbone assignment applied to two large proteins: intrinsically disordered 441-residue Tau and a 509-residue globular bacteriophytochrome fragment. The approach will be valuable in a multitude of chemistry applications, where NMR experiments are compromised by the homonuclear scalar coupling.

Deciphering the molecular mechanism of FLT3 resistance mutations.

FMS-like tyrosine kinase 3 (FLT3) has been found to be mutated in ~ 30% of acute myeloid leukaemia patients. Small-molecule inhibitors targeting FLT3 that are currently approved or still undergoing clinical trials are subject to drug resistance due to FLT3 mutations. How these mutations lead to drug resistance is hitherto poorly understood. Herein, we studied the molecular mechanism of the drug resistance mutations D835N, Y842S and M664I, which confer resistance against the most advanced inhibitors, quizartinib and PLX3397 (pexidartinib), using enzyme kinetics and computer simulations. In vitro kinase assays were performed to measure the comparative catalytic activity of the native protein and the mutants, using a bacterial expression system developed to this aim. Our results reveal that the differential drug sensitivity profiles can be rationalised by the dynamics of the protein-drug interactions and perturbation of the intraprotein contacts upon mutations. Drug binding induced a single conformation in the native protein, whereas multiple conformations were observed otherwise (in the mutants or in the absence of drugs). The end-point kinetics measurements indicated that the three resistant mutants conferred catalytic activity that is at least as high as that of the reference without such mutations. Overall, our calculations and measurements suggest that the structural dynamics of the drug-resistant mutants that affect the active state and the increased conformational freedom of the remaining inactive drug-bound population are the two major factors that contribute to drug resistance in FLT3 harbouring cancer cells. Our results explain the mechanism of drug resistance mutations and can aid to the design of more effective tyrosine kinase inhibitors.

Molecular simulation of peptides coming of age: Accurate prediction of folding, dynamics and structures.

The application of molecular dynamics simulations to study the folding and dynamics of peptides has attracted a lot of interest in the last couple of decades. Following the successful prediction of the folding of several proteins using molecular simulation, foldable peptides emerged as a favourable system mainly due to their application in improving protein structure prediction methods and in drug design studies. However, their performance is inherently linked to the accuracy of the empirical force fields used in the simulations, whose optimisation and validation is of paramount importance. Here we review the most important findings in the field of molecular peptide simulations and highlight the significant advancements made over the last twenty years. Special reference is made on the simulation of disordered peptides and the remaining challenge to find a force field able to describe accurately their conformational landscape.

The catalytic activity of Abl1 single and compound mutations: Implications for the mechanism of drug resistance mutations in chronic myeloid leukaemia.

BACKGROUND: Abl1 is a protein tyrosine kinase whose aberrant activation due to mutations is the culprit of several cancers, most notably chronic myeloid leukaemia. Several Abl1 inhibitors are used as anti-cancer drugs. Unfortunately, drug resistance limits their effectiveness. The main cause for drug resistance is mutations in the kinase domain (KD) of Abl1 that evolve in patients. The T315I mutation confers resistance against all clinically-available inhibitors except ponatinib. Resistance to ponatinib can develop by compound (double) mutations. METHODS: Kinetic measurements of the KD of Abl1 and its mutants were carried out to examine their catalytic activity. Specifically, mutants that lead to drug resistance against ponatinib were considered. Molecular dynamics simulations and multiple sequence analysis were used for explanation of the experimental findings. RESULTS: The catalytic efficiency of the T315I pan-resistance mutant is more than two times lower than that of the native KD. All ponatinib resistant mutations restore the catalytic efficiency of the enzyme. Two of them (G250E/T315I and Y253H/E255V) have a catalytic efficiency that is more than five times that of the native KD. CONCLUSIONS: The measurements and analysis suggest that resistance is at least partially due to the development of a highly efficient kinase through subsequent mutations. The simulations highlight modifications in two structurally important regions of Abl1, the activation and phosphate binding loops, upon mutations. GENERAL SIGNIFICANCE: Experimental and computational methods were used together to explain how mutations in the kinase domain of Abl1 lead to resistance against the most advanced drug currently in use to treat chronic myeloid leukaemia.

Folding Molecular Dynamics Simulation of a gp41-Derived Peptide Reconcile Divergent Structure Determinations.

T-20 peptide is the first FDA-approved fusion inhibitor against AIDS/HIV-1 gp41 protein. Part of it, the gp41[659-671] peptide, that contains the complete epitope for the neutralizing 2F5 monoclonal antibody, has been found experimentally in a number of divergent structures. Herein, we attempt to reconcile them by using unbiased large-scale all-atom molecular dynamics folding simulations. We show that our approach can successfully capture the peptide's heterogeneity and reach each and every experimentally determined conformation in sub-angstrom accuracy, whilst preserving the peptide's disordered nature. Our analysis also unveils that the minor refinements within the AMBER99SB family of force fields can lead to appreciable differences in the predicted conformational stability arising from subtle differences in the helical- and ß-region of the Ramachandran plot. Our work underlines the contribution of molecular dynamics simulation in structurally characterizing pharmacologically important peptides of ambiguous structure.

Novel FRET-substrates of Rhizomucor pusillus rennin: Activity and mechanistic studies.

The development of sensitive, easy and reliable methods for the determination of Rhizomucor pusillus rennin (MPR) activity, in free and in immobilized form, along with the elucidation of the mechanism of action, represent challenges for the widespread use of the enzyme in industrial cheese production. These could be accomplished by using highly specific and sensitive substrates, as well as direct assay methods. We designed and synthesized novel substrates based on Fluorescence Resonance Energy Transfer (FRET) for the MPR by employing computational simulation techniques and peptide synthesis in liquid phase. Three FRET-substrates (Abz-GFY-pNA, Abz-SFY-pNA and Abz-GFI-pNA) were found active, while the Abz-GFY-pNA showed the highest reliability, sensitivity and specificity among them. Subsequently, a novel mechanism of MPR action was elucidated, with the development of novel methods for assaying activity in free and immobilized form, which both may contribute in the wider use of rennin in cheese production and other biotechnological applications.

On the foldability of tryptophan-containing tetra- and pentapeptides: an exhaustive molecular dynamics study.

Short peptides serve as minimal model systems to decipher the determinants of foldability due to their simplicity arising from their smaller size, their ability to echo protein-like structural characteristics, and their direct implication in force field validation. Here, we describe an effort to identify small peptides that can still form stable structures in aqueous solutions. We followed the in silico folding of a selected set of 8640 tryptophan-containing tetra- and pentapeptides through 15 210 molecular dynamics simulations amounting to a total of 272.46 µs using explicit representation of the solute and full treatment of the electrostatics. The evaluation and sorting of peptides is achieved through scoring functions, which include terms based on interatomic vector distances, atomic fluctuations, and rmsd matrices between successive frames of a trajectory. Highly scored peptides are studied further via successive simulation rounds of increasing simulation length and using different empirical force fields. Our method suggested only a handful of peptides with strong foldability prognosis. The discrepancies between the predictions of the various force fields for such short sequences are also extensively discussed. We conclude that the vast majority of such short peptides do not adopt significantly stable structures in water solutions, at least based on our computational predictions. The present work can be utilized in the rational design and engineering of bioactive peptides with desired molecular properties.

Using J-coupling constants for force field validation: application to hepta-alanine.

A computational solution to the protein folding problem is the holy grail of biomolecular simulation and of the corresponding force fields. The complexity of the systems used for folding simulations precludes a direct feedback between the simulations and the force fields, thus necessitating the study of simpler systems with sufficient experimental data to allow force field optimization and validation. Recent studies on short polyalanine peptides of increasing length (up to penta-alanine) indicated the presence of a systematic deviation between the experimental (NMR-derived) J-couplings and the great majority of biomolecular force fields, with the χ(2) values for even the best-performing force fields being in the 1.4-1.8 range. Here we show that by increasing the number of residues to seven and by achieving convergence through an increase of the simulation time to 2 µs, we can identify one force field (the AMBER99SB force field, out of the three force fields studied) which when compared with the experimental J-coupling data (and for a specific set of Karplus equation parameters and estimated J-coupling errors previously used in the literature) gave a value of χ(2) = 0.99, indicating that full statistical consistency between experiment and simulation is feasible. However, and as a detailed analysis of the effects of estimated errors shows, the χ(2) values may be unsuitable as indicators of the goodness of fit of the various biomolecular force fields.