NMR tools to detect protein allostery.

Allostery is a fundamental mechanism of cellular homeostasis by intra-protein communication between distinct functional sites. It is an internal process of proteins to steer interactions not only with each other but also with other biomolecules such as ligands, lipids, and nucleic acids. In addition, allosteric regulation is particularly important in enzymatic activities. A major challenge in structural and molecular biology today is unraveling allosteric sites in proteins, to elucidate the detailed mechanism of allostery and the development of allosteric drugs. Here we summarize the recently developed tools and approaches which enable the elucidation of regulatory hotspots and correlated motion in biomolecules, focusing primarily on solution-state nuclear magnetic resonance spectroscopy (NMR). These tools open an avenue towards a rational understanding of the mechanism of allostery and provide essential information for the design of allosteric drugs.

The 100-protein NMR spectra dataset: A resource for biomolecular NMR data analysis.

Multidimensional NMR spectra are the basis for studying proteins by NMR spectroscopy and crucial for the development and evaluation of methods for biomolecular NMR data analysis. Nevertheless, in contrast to derived data such as chemical shift assignments in the BMRB and protein structures in the PDB databases, this primary data is in general not publicly archived. To change this unsatisfactory situation, we present a standardized set of solution NMR data comprising 1329 2-4-dimensional NMR spectra and associated reference (chemical shift assignments, structures) and derived (peak lists, restraints for structure calculation, etc.) annotations. With the 100-protein NMR spectra dataset that was originally compiled for the development of the ARTINA deep learning-based spectra analysis method, 100 protein structures can be reproduced from their original experimental data. The 100-protein NMR spectra dataset is expected to help the development of computational methods for NMR spectroscopy, in particular machine learning approaches, and enable consistent and objective comparisons of these methods.

Ultrafast Fragment Screening Using Photo-Hyperpolarized (CIDNP) NMR.

While nuclear magnetic resonance (NMR) is regarded as a reference in fragment-based drug design, its implementation in a high-throughput manner is limited by its lack of sensitivity resulting in long acquisition times and high micromolar sample concentrations. Several hyperpolarization approaches could, in principle, improve the sensitivity of NMR also in drug research. However, photochemically induced dynamic nuclear polarization (photo-CIDNP) is the only method that is directly applicable in aqueous solution and agile for scalable implementation using off-the-shelf hardware. With the use of photo-CIDNP, this work demonstrates the detection of weak binders in the millimolar affinity range using low micromolar concentrations down to 5 µM of ligand and 2 µM of target, thereby exploiting the photo-CIDNP-induced polarization twice: (i) increasing the signal-to-noise by one to two orders in magnitude and (ii) polarization-only of the free non-bound molecule allowing identification of binding by polarization quenching, yielding another factor of hundred in time when compared with standard techniques. The interaction detection was performed with single-scan NMR experiments of a duration of 2 to 5 s. Taking advantage of the readiness of photo-CIDNP setup implementation, an automated flow-through platform was designed to screen samples at a screening rate of 1500 samples per day. Furthermore, a 212 compounds photo-CIDNP fragment library is presented, opening an avenue toward a comprehensive fragment-based screening method.

Multi-Dimensional Structure and Dynamics Landscape of Proteins in Mammalian Cells Revealed by In-Cell NMR.

Governing function, half-life and subcellular localization, the 3D structure and dynamics of proteins are in nature constantly changing in a tightly regulated manner to fulfill the physiological and adaptive requirements of the cells. To find evidence for this hypothesis, we applied in-cell NMR to three folded model proteins and propose that the splitting of cross peaks constitutes an atomic fingerprint of distinct structural states that arise from multiple target binding co-existing inside mammalian cells. These structural states change upon protein loss of function or subcellular localisation into distinct cell compartments. In addition to peak splitting, we observed NMR signal intensity attenuations indicative of transient interactions with other molecules and dynamics on the microsecond to millisecond time scale.

Protein structure determination in human cells by in-cell NMR and a reporter system to optimize protein delivery or transexpression.

Most experimental methods for structural biology proceed in vitro and therefore the contribution of the intracellular environment on protein structure and dynamics is absent. Studying proteins at atomic resolution in living mammalian cells has been elusive due to the lack of methodologies. In-cell nuclear magnetic resonance spectroscopy (in-cell NMR) is an emerging technique with the power to do so. Here, we improved current methods of in-cell NMR by the development of a reporter system that allows monitoring the delivery of exogenous proteins into mammalian cells, a process that we called here "transexpression". The reporter system was used to develop an efficient protocol for in-cell NMR which enables spectral acquisition with higher quality for both disordered and folded proteins. With this method, the 3D atomic resolution structure of the model protein GB1 in human cells was determined with a backbone root-mean-square deviation (RMSD) of 1.1 Å.

Atomic resolution protein allostery from the multi-state structure of a PDZ domain.

Recent methodological advances in solution NMR allow the determination of multi-state protein structures and provide insights into structurally and dynamically correlated protein sites at atomic resolution. This is demonstrated in the present work for the well-studied PDZ2 domain of protein human tyrosine phosphatase 1E for which protein allostery had been predicted. Two-state protein structures were calculated for both the free form and in complex with the RA-GEF2 peptide using the exact nuclear Overhauser effect (eNOE) method. In the apo protein, an allosteric conformational selection step comprising almost 60% of the domain was detected with an "open" ligand welcoming state and a "closed" state that obstructs the binding site by changing the distance between the ß-sheet 2, α-helix 2, and sidechains of residues Lys38 and Lys72. The observed induced fit-type apo-holo structural rearrangements are in line with the previously published evolution-based analysis covering ~25% of the domain with only a partial overlap with the protein allostery of the open form. These presented structural studies highlight the presence of a dedicated highly optimized and complex dynamic interplay of the PDZ2 domain owed by the structure-dynamics landscape.

Optimization and validation of multi-state NMR protein structures using structural correlations.

Recent advances in the field of protein structure determination using liquid-state NMR enable the elucidation of multi-state protein conformations that can provide insight into correlated and non-correlated protein dynamics at atomic resolution. So far, NMR-derived multi-state structures were typically evaluated by means of visual inspection of structure superpositions, target function values that quantify the violation of experimented restraints and root-mean-square deviations that quantify similarity between conformers. As an alternative or complementary approach, we present here the use of a recently introduced structural correlation measure, PDBcor, that quantifies the clustering of protein states as an additional measure for multi-state protein structure analysis. It can be used for various assays including the validation of experimental distance restraints, optimization of the number of protein states, estimation of protein state populations, identification of key distance restraints, NOE network analysis and semiquantitative analysis of the protein correlation network. We present applications for the final quality analysis stages of typical multi-state protein structure calculations.

An improved, time-efficient approach to extract accurate distance restraints for NMR2 structure calculation.

Exact nuclear Overhauser enhancement (eNOE) yields highly accurate, ensemble averaged 1H-1H distance restraints with an accuracy of up to 0.1 Šfor the multi-state structure determination of proteins as well as for nuclear magnetic resonance molecular replacement (NMR2) to determine the structure of the protein-ligand interaction site in a time-efficient manner. However, in the latter application, the acquired eNOEs lack the obtainable precision of 0.1 Šbecause of the asymmetrical nature of the filtered nuclear Overhauser enhancement spectroscopy (NOESY) experiment used in NMR2. This error is further propagated to the eNOE equations used to fit and extract the distance restraints. In this work, a new analysis method is proposed to obtain inter-molecular distance restraints from the filtered NOESY spectrum more accurately and intuitively by dividing the NOE cross peak by the corresponding diagonal peak of the ligand. The method termed diagonal-normalised eNOEs was tested on the data acquired by on the complex of PIN1 and a small, weak-binding phenylimidazole fragment. NMR2 calculations performed using the distances derived from diagonal-normalised eNOEs yielded the right orientation of the fragment in the binding pocket and produced a structure that more closely resembles the benchmark X-ray structure (2XP6) with an average heavy-atom root-mean-square deviation (RMSD) of 1.681 Šwith respect to it, when compared to the one produced with traditional NMR2 with an average heavy atom RMSD of 3.628 Å. This is attributed to the higher precision of the evaluated distance restraints.

PDBcor: An automated correlation extraction calculator for multi-state protein structures.

Allostery and correlated motion are key elements linking protein dynamics with the mechanisms of action of proteins. Here, we present PDBCor, an automated and unbiased method for the detection and analysis of correlated motions from experimental multi-state protein structures. It uses torsion angle and distance statistics and does not require any structure superposition. Clustering of protein conformers allows us to extract correlations in the form of mutual information based on information theory. With PDBcor, we elucidated correlated motion in the WW domain of PIN1, the protein GB3, and the enzyme cyclophilin, in line with reported findings. Correlations extracted with PDBcor can be utilized in subsequent assays including nuclear magnetic resonance (NMR) multi-state structure optimization and validation. As a guide for the interpretation of PDBcor results, we provide a series of protein structure ensembles that exhibit different levels of correlation, including non-correlated, locally correlated, and globally correlated ensembles.

Disease-Associated Tau Phosphorylation Hinders Tubulin Assembly within Tau Condensates.

Cellular condensation of intrinsically disordered proteins (IDPs) through liquid-liquid phase separation (LLPS) allows dynamic compartmentalization and regulation of biological processes. The IDP tau, which promotes the assembly of microtubules and is hyperphosphorylated in Alzheimer's disease, undergoes LLPS in solution and on the surface of microtubules. Little is known, however, about the influence of tau phosphorylation on its ability to nucleate microtubule bundles in conditions of tau LLPS. Herein, we show that unmodified tau as well as tau phosphorylated at disease-associated epitopes condense into liquid-like droplets. Although tubulin partitioned into and reached high concentrations inside all tau droplets, it was unable to grow into microtubules form the inside of droplets formed by tau phosphorylated at the AT180 epitope (T231/S235). In contrast, neither phosphorylation of tau in the repeat domain nor at its tyrosine residues inhibited the assembly of tubulin from tau droplets. Because LLPS of IDPs has been shown to promote different types of cytoskeletal assembly, our study suggests that IDP phosphorylation might be a broadly used mechanism for the modulation of condensate-mediated cytoskeletal assembly.

Protein Allostery at Atomic Resolution.

Protein allostery is a phenomenon involving the long range coupling between two distal sites in a protein. In order to elucidate allostery at atomic resoluion on the ligand-binding WW domain of the enzyme Pin1, multistate structures were calculated from exact nuclear Overhauser effect (eNOE). In its free form, the protein undergoes a microsecond exchange between two states, one of which is predisposed to interact with its parent catalytic domain. In presence of the positive allosteric ligand, the equilibrium between the two states is shifted towards domain-domain interaction, suggesting a population shift model. In contrast, the allostery-suppressing ligand decouples the side-chain arrangement at the inter-domain interface thereby reducing the inter-domain interaction. As such, this mechanism is an example of dynamic allostery. The presented distinct modes of action highlight the power of the interplay between dynamics and function in the biological activity of proteins.

Evaluation of the methoxy-X04 derivative BSC4090 for diagnosis of prodromal and early Alzheimer's disease from bioptic olfactory mucosa.

Alzheimer's disease (AD) pathology precedes the onset of clinical symptoms by several decades. Thus, biomarkers are required to identify prodromal disease stages to allow for the early and effective treatment. The methoxy-X04-derivative BSC4090 is a fluorescent ligand which was designed to target neurofibrillary tangles in AD. BSC4090 staining was previously detected in post-mortem brains and olfactory mucosa derived from AD patients. We tested BSC4090 as a potential diagnostic marker of prodromal and early AD using olfactory mucosa biopsies from 12 individuals with AD, 13 with mild cognitive impairment (MCI), and 10 cognitively normal (CN) controls. Receiver-operating curve analysis revealed areas under the curve of 0.78 for AD versus CN and of 0.86 for MCI due to AD versus MCI of other causes. BSC4090 labeling correlated significantly with cerebrospinal fluid levels of tau protein phosphorylated at T181. Using NMR spectroscopy, we find that BSC4090 binds to fibrillar and pre-fibrillar but not to monomeric tau. Thus, BSC4090 may be an interesting candidate to detect AD at the early disease stages.

Mapping interactions with the chaperone network reveals factors that protect against tau aggregation.

A network of molecular chaperones is known to bind proteins ('clients') and balance their folding, function and turnover. However, it is often unclear which chaperones are critical for selective recognition of individual clients. It is also not clear why these key chaperones might fail in protein-aggregation diseases. Here, we utilized human microtubule-associated protein tau (MAPT or tau) as a model client to survey interactions between ~30 purified chaperones and ~20 disease-associated tau variants (~600 combinations). From this large-scale analysis, we identified human DnaJA2 as an unexpected, but potent, inhibitor of tau aggregation. DnaJA2 levels were correlated with tau pathology in human brains, supporting the idea that it is an important regulator of tau homeostasis. Of note, we found that some disease-associated tau variants were relatively immune to interactions with chaperones, suggesting a model in which avoiding physical recognition by chaperone networks may contribute to disease.

The Binding Mode of a Tau Peptide with Tubulin.

The microtubule-associated protein Tau promotes the polymerization of tubulin and modulates the function of microtubules. As a consequence of the dynamic nature of the Tau-tubulin interaction, the structural basis of this complex has remained largely elusive. By using NMR methods optimized for ligand-receptor interactions in combination with site-directed mutagenesis we demonstrate that the flanking domain downstream of the four microtubule-binding repeats of Tau binds competitively to a site on the α-tubulin surface. The binding process is complex, involves partial coupling of different interacting regions, and is modulated by phosphorylation at Y394 and S396. This study strengthens the hypothesis of an intimate relationship between Tau phosphorylation and tubulin binding and highlights the power of the INPHARMA NMR method to characterize the interaction of peptides derived from intrinsically disordered proteins with their molecular partners.

Multivalent cross-linking of actin filaments and microtubules through the microtubule-associated protein Tau.

Microtubule-associated proteins regulate microtubule dynamics, bundle actin filaments, and cross-link actin filaments with microtubules. In addition, aberrant interaction of the microtubule-associated protein Tau with filamentous actin is connected to synaptic impairment in Alzheimer's disease. Here we provide insight into the nature of interaction between Tau and actin filaments. We show that Tau uses several short helical segments to bind in a dynamic, multivalent process to the hydrophobic pocket between subdomains 1 and 3 of actin. Although a single Tau helix is sufficient to bind to filamentous actin, at least two, flexibly linked helices are required for actin bundling. In agreement with a structural model of Tau repeat sequences in complex with actin filaments, phosphorylation at serine 262 attenuates binding of Tau to filamentous actin. Taken together the data demonstrate that bundling of filamentous actin and cross-linking of the cellular cytoskeleton depend on the metamorphic and multivalent nature of microtubule-associated proteins.

Structural Impact of Tau Phosphorylation at Threonine 231.

Phosphorylation of the microtubule-associated protein Tau influences the assembly and stabilization of microtubules and is deregulated in several neurodegenerative diseases. The high flexibility of Tau, however, has prevented an atomic-level description of its phosphorylation-induced structural changes. Employing an extensive set of distance and orientational restraints together with a novel ensemble calculation approach, we determined conformational ensembles of Tau fragments in the non-phosphorylated state and, when phosphorylated at T231/S235 or T231/S235/S237/S238, four important sites of phosphorylation in Alzheimer disease. Comparison of the molecular ensembles showed that phosphorylation of the regulatory T231 does not perturb the backbone conformation of the proximal microtubule-binding (225)KVAVVR(230) motif. Instead, phosphorylated T231 selectively engages in a salt bridge with R230 that can compete with the formation of intermolecular salt bridges to tubulin. Our study provides an ensemble description which will be useful for the analysis of conformational transitions in Tau and other intrinsically disordered proteins.

Folding of the Tau Protein on Microtubules.

Microtubules are regulated by microtubule-associated proteins. However, little is known about the structure of microtubule-associated proteins in complex with microtubules. Herein we show that the microtubule-associated protein Tau, which is intrinsically disordered in solution, locally folds into a stable structure upon binding to microtubules. While Tau is highly flexible in solution and adopts a ß-sheet structure in amyloid fibrils, in complex with microtubules the conserved hexapeptides at the beginning of the Tau repeats two and three convert into a hairpin conformation. Thus, binding to microtubules stabilizes a unique conformation in Tau.

Tau stabilizes microtubules by binding at the interface between tubulin heterodimers.

The structure, dynamic behavior, and spatial organization of microtubules are regulated by microtubule-associated proteins. An important microtubule-associated protein is the protein Tau, because its microtubule interaction is impaired in the course of Alzheimer's disease and several other neurodegenerative diseases. Here, we show that Tau binds to microtubules by using small groups of evolutionary conserved residues. The binding sites are formed by residues that are essential for the pathological aggregation of Tau, suggesting competition between physiological interaction and pathogenic misfolding. Tau residues in between the microtubule-binding sites remain flexible when Tau is bound to microtubules in agreement with a highly dynamic nature of the Tau-microtubule interaction. By binding at the interface between tubulin heterodimers, Tau uses a conserved mechanism of microtubule polymerization and, thus, regulation of axonal stability and cell morphology.

Phosphorylation of human Tau protein by microtubule affinity-regulating kinase 2.

Tau protein plays an important role in neuronal physiology and Alzheimer's neurodegeneration. Its abilities to aggregate abnormally, to bind to microtubules (MTs), and to promote MT assembly are all influenced by phosphorylation. Phosphorylation of serine residues in the KXGS motifs of Tau's repeat domain, crucial for MT interactions and aggregation, is facilitated most efficiently by microtubule-associated protein/microtubule affinity-regulating kinases (MARKs). Here we applied high-resolution nuclear magnetic resonance analysis to study the kinetics of phosphorylation of Tau by MARK2 and its impact on the structure and microtubule binding of Tau. We demonstrate that MARK2 binds to the N-terminal tail of Tau and selectively phosphorylates three major and five minor serine residues in the repeat domain and C-terminal tail. Structural changes induced by phosphorylation of Tau by MARK2 are highly localized in the proximity of the phosphorylation site and do not affect the global conformation, in contrast to phosphorylation in the proline-rich region. Furthermore, single-residue analysis of binding of Tau to MTs provides support for a model in which Tau's hot spots of MT interaction bind independently of each other and are differentially affected by phosphorylation.