High-resolution structural information of membrane-bound α-synuclein provides insight into the MoA of the anti-Parkinson drug UCB0599.

α-synuclein (αS) is an intrinsically disordered protein whose functional ambivalence and protein structural plasticity are iconic. Coordinated protein recruitment ensures proper vesicle dynamics at the synaptic cleft, while deregulated oligomerization on cellular membranes contributes to cell damage and Parkinson's disease (PD). Despite the protein's pathophysiological relevance, structural knowledge is limited. Here, we employ NMR spectroscopy and chemical cross-link mass spectrometry on 14N/15N-labeled αS mixtures to provide for the first time high-resolution structural information of the membrane-bound oligomeric state of αS and demonstrate that in this state, αS samples a surprisingly small conformational space. Interestingly, the study locates familial Parkinson's disease mutants at the interface between individual αS monomers and reveals different oligomerization processes depending on whether oligomerization occurs on the same membrane surface (cis) or between αS initially attached to different membrane particles (trans). The explanatory power of the obtained high-resolution structural model is used to help determine the mode-of-actionof UCB0599. Here, it is shown that the ligand changes the ensemble of membrane-bound structures, which helps to explain the success this compound, currently being tested in Parkinson's disease patients in a phase 2 trial, has had in animal models of PD.

Decorating phenylalanine side-chains with triple labeled 13C/19F/2H isotope patterns.

We present an economic and straightforward method to introduce 13C-19F spin systems into the deuterated aromatic side chains of phenylalanine as reporters for various protein NMR applications. The method is based on the synthesis of [4-13C, 2,3,5,6-2H4] 4-fluorophenylalanine from the commercially available isotope sources [2-13C] acetone and deuterium oxide. This compound is readily metabolized by standard Escherichia coli overexpression in a glyphosate-containing minimal medium, which results in high incorporation rates in the corresponding target proteins.

Synthesis of a 13C-methylene-labeled isoleucine precursor as a useful tool for studying protein side-chain interactions and dynamics.

In this study, we present the synthesis and incorporation of a metabolic isoleucine precursor compound for selective methylene labeling. The utility of this novel α-ketoacid isotopologue is shown by incorporation into the protein Brd4-BD1, which regulates gene expression by binding to acetylated histones. High quality single quantum 13C-1 H-HSQC were obtained, as well as triple quantum HTQC spectra, which are superior in terms of significantly increased 13C-T2 times. Additionally, large chemical shift perturbations upon ligand binding were observed. Our study thus proves the great sensitivity of this precursor as a reporter for side-chain dynamic studies and for investigations of CH-π interactions in protein-ligand complexes.

13 Cß-Valine and 13 Cγ-Leucine Methine Labeling To Probe Protein Ligand Interaction.

Precise information regarding the interaction between proteins and ligands at molecular resolution is crucial for effectively guiding the optimization process from initial hits to lead compounds in early stages of drug development. In this study, we introduce a novel aliphatic side chain isotope-labeling scheme to directly probe interactions between ligands and aliphatic sidechains using NMR techniques. To demonstrate the applicability of this method, we selected a set of Brd4-BD1 binders and analyzed 1 H chemical shift perturbation resulting from CH-π interaction of Hß -Val and Hγ -Leu as CH donors with corresponding ligand aromatic moieties as π acceptors.

Ligand 1 H NMR Chemical Shifts as Accurate Reporters for Protein-Ligand Binding Interfaces in Solution.

The availability of high-resolution 3D structural information is crucial for investigating guest-host systems across a wide range of fields. In the context of drug discovery, the information is routinely used to establish and validate structure-activity relationships, grow initial hits from screening campaigns, and to guide molecular docking. For the generation of protein-ligand complex structural information, X-ray crystallography is the experimental method of choice, however, with limited information on protein flexibility. An experimentally verified structural model of the binding interface in the native solution-state would support medicinal chemists in their molecular design decisions. Here we demonstrate that protein-bound ligand 1 H NMR chemical shifts are highly sensitive and accurate probes for the immediate chemical environment of protein-ligand interfaces. By comparing the experimental ligand 1 H chemical shift values with those computed from the X-ray structure using quantum mechanics methodology, we identify significant disagreements for parts of the ligand between the two experimental techniques. We show that quantum mechanics/molecular mechanics (QM/MM) molecular dynamics (MD) ensembles can be used to refine initial X-ray co-crystal structures resulting in a better agreement with experimental 1 H ligand chemical shift values. Overall, our findings highlight the usefulness of ligand 1 H NMR chemical shift information in combination with a QM/MM MD workflow for generating protein-ligand ensembles that accurately reproduce solution structural data.

Molecular basis of F-actin regulation and sarcomere assembly via myotilin.

Sarcomeres, the basic contractile units of striated muscle cells, contain arrays of thin (actin) and thick (myosin) filaments that slide past each other during contraction. The Ig-like domain-containing protein myotilin provides structural integrity to Z-discs-the boundaries between adjacent sarcomeres. Myotilin binds to Z-disc components, including F-actin and α-actinin-2, but the molecular mechanism of binding and implications of these interactions on Z-disc integrity are still elusive. To illuminate them, we used a combination of small-angle X-ray scattering, cross-linking mass spectrometry, and biochemical and molecular biophysics approaches. We discovered that myotilin displays conformational ensembles in solution. We generated a structural model of the F-actin:myotilin complex that revealed how myotilin interacts with and stabilizes F-actin via its Ig-like domains and flanking regions. Mutant myotilin designed with impaired F-actin binding showed increased dynamics in cells. Structural analyses and competition assays uncovered that myotilin displaces tropomyosin from F-actin. Our findings suggest a novel role of myotilin as a co-organizer of Z-disc assembly and advance our mechanistic understanding of myotilin's structural role in Z-discs.

Semisynthesis of segmentally isotope-labeled and site-specifically palmitoylated CD44 cytoplasmic tail.

CD44, a ubiquitously expressed transmembrane receptor, plays a crucial role in cell growth, migration, and tumor progression. Dimerization of CD44 is a key event in signal transduction and has emerged as a potential target for anti-tumor therapies. Palmitoylation, a posttranslational modification, disrupts CD44 dimerization and promotes CD44 accumulation in ordered membrane domains. However, the effects of palmitoylation on the structure and dynamics of CD44 at atomic resolution remain poorly understood. Here, we present a semisynthetic approach combining solid-phase peptide synthesis, recombinant expression, and native chemical ligation to investigate the impact of palmitoylation on the cytoplasmic domain (residues 669-742) of CD44 (CD44ct) by NMR spectroscopy. A segmentally isotope-labeled and site-specifically palmitoylated CD44 variant enabled NMR studies, which revealed chemical shift perturbations and indicated local and long-range conformational changes induced by palmitoylation. The long-range effects suggest altered intramolecular interactions and potential modulation of membrane association patterns. Semisynthetic, palmitoylated CD44ct serves as the basis for studying CD44 clustering, conformational changes, and localization within lipid rafts, and could be used to investigate its role as a tumor suppressor and to explore its therapeutic potential.

Non-uniform sampling of similar NMR spectra and its application to studies of the interaction between alpha-synuclein and liposomes.

The accelerated acquisition of multidimensional NMR spectra using sparse non-uniform sampling (NUS) has been widely adopted in recent years. The key concept in NUS is that a major part of the data is omitted during measurement, and then reconstructed using, for example, compressed sensing (CS) methods. CS requires spectra to be compressible, that is, they should contain relatively few "significant" points. The more compressible the spectrum, the fewer experimental NUS points needed in order for it to be accurately reconstructed. In this paper we show that the CS processing of similar spectra can be enhanced by reconstructing only the differences between them. Accurate reconstruction can be obtained at lower sampling levels as the difference is sparser than the spectrum itself. In many situations this method is superior to "conventional" compressed sensing. We exemplify the concept of "difference CS" with one such case-the study of alpha-synuclein binding to liposomes and its dependence on temperature. To obtain information on temperature-dependent transitions between different states, we need to acquire several dozen spectra at various temperatures, with and without the presence of liposomes. Our detailed investigation reveals that changes in the binding modes of the alpha-synuclein ensemble are not only temperature-dependent but also show non-linear behavior in their transitions. Our proposed CS processing approach dramatically reduces the number of NUS points required and thus significantly shortens the experimental time.

Cosolute modulation of protein oligomerization reactions in the homeostatic timescale.

Protein oligomerization processes are widespread and of crucial importance to understand degenerative diseases and healthy regulatory pathways. One particular case is the homo-oligomerization of folded domains involving domain swapping, often found as a part of the protein homeostasis in the crowded cytosol, composed of a complex mixture of cosolutes. Here, we have investigated the effect of a plethora of cosolutes of very diverse nature on the kinetics of a protein dimerization by domain swapping. In the absence of cosolutes, our system exhibits slow interconversion rates, with the reaction reaching the equilibrium within the average protein homeostasis timescale (24-48 h). In the presence of crowders, though, the oligomerization reaction in the same time frame will, depending on the protein's initial oligomeric state, either reach a pure equilibrium state or get kinetically trapped into an apparent equilibrium. Specifically, when the reaction is initiated from a large excess of dimer, it becomes unsensitive to the effect of cosolutes and reaches the same equilibrium populations as in the absence of cosolute. Conversely, when the reaction starts from a large excess of monomer, the reaction during the homeostatic timescale occurs under kinetic control, and it is exquisitely sensitive to the presence and nature of the cosolute. In this scenario (the most habitual case in intracellular oligomerization processes), the effect of cosolutes on the intermediate conformation and diffusion-mediated encounters will dictate how the cellular milieu affects the domain-swapping reaction.

Hyperphosphorylation of Human Osteopontin and Its Impact on Structural Dynamics and Molecular Recognition.

Protein phosphorylation is an abundant post-translational modification (PTM) and an essential modulator of protein functionality in living cells. Intrinsically disordered proteins (IDPs) are particular targets of PTM protein kinases due to their involvement in fundamental protein interaction networks. Despite their dynamic nature, IDPs are far from having random-coil conformations but exhibit significant structural heterogeneity. Changes in the molecular environment, most prominently in the form of PTM via phosphorylation, can modulate these structural features. Therefore, how phosphorylation events can alter conformational ensembles of IDPs and their interactions with binding partners is of great interest. Here we study the effects of hyperphosphorylation on the IDP osteopontin (OPN), an extracellular target of the Fam20C kinase. We report a full characterization of the phosphorylation sites of OPN using a combined nuclear magnetic resonance/mass spectrometry approach and provide evidence for an increase in the local flexibility of highly phosphorylated regions and the ensuing overall structural elongation. Our study emphasizes the simultaneous importance of electrostatic and hydrophobic interactions in the formation of compact substates in IDPs and their relevance for molecular recognition events.

Binding Mode Characterization of Osteopontin on Hydroxyapatite by Solution NMR Spectroscopy.

Extracellular matrix glycoproteins play a major role in bone mineralization and modulation of osteogenesis. Among these, the intrinsically disordered protein osteopontin (OPN) is associated with the inhibition of formation, growth and proliferation of the bone mineral hydroxyapatite (HAP). Furthermore, post-translational modifications like phosphorylation can alter conformations and interaction properties of intrinsically disordered proteins (IDPs). Therefore, the actual interaction of OPN with a HAP surface on an atomic level and how this interaction is affected by phosphorylation is of great interest. Here, we study the interaction of full-length OPN on the surface of suspended HAP nanoparticles by solution NMR spectroscopy. We report the binding modes of this IDP and provide evidence for the influence of hyperphosphorylation on the binding character and an explanation for the differing roles in biomineralization. Our study moreover presents an easy and suitable option to measure interaction of nanoparticles in a stable suspension with full-length proteins.

Temperature as an Extra Dimension in Multidimensional Protein NMR Spectroscopy.

NMR spectroscopy is a particularly informative method for studying protein structures and dynamics in solution; however, it is also one of the most time-consuming. Modern approaches to biomolecular NMR spectroscopy are based on lengthy multidimensional experiments, the duration of which grows exponentially with the number of dimensions. The experimental time may even be several days in the case of 3D and 4D spectra. Moreover, the experiment often has to be repeated under several different conditions, for example, to measure the temperature-dependent effects in a spectrum (temperature coefficients (TCs)). Herein, a new approach that involves joint sampling of indirect evolution times and temperature is proposed. This allows TCs to be measured through 3D spectra in even less time than that needed to acquire a single spectrum by using the conventional approach. Two signal processing methods that are complementary, in terms of sensitivity and resolution, 1) dividing data into overlapping subsets followed by compressed sensing reconstruction, and 2) treating the complete data set with a variant of the Radon transform, are proposed. The temperature-swept 3D HNCO spectra of two intrinsically disordered proteins, osteopontin and CD44 cytoplasmic tail, show that this new approach makes it possible to determine TCs and their non-linearities effectively. Non-linearities, which indicate the presence of a compact state, are particularly interesting. The complete package of data acquisition and processing software for this new approach are provided.

Using Cross-Correlated Spin Relaxation to Characterize Backbone Dihedral Angle Distributions of Flexible Protein Segments.

Crucial to the function of proteins is their existence as conformational ensembles sampling numerous and structurally diverse substates. Despite this widely accepted notion there is still a high demand for meaningful and reliable approaches to characterize protein ensembles in solution. As it is usually conducted in solution, NMR spectroscopy offers unique possibilities to address this challenge. Particularly, cross-correlated relaxation (CCR) effects have long been established to encode both protein structure and dynamics in a compelling manner. However, this wealth of information often limits their use in practice as structure and dynamics might prove difficult to disentangle. Using a modern Maximum Entropy (MaxEnt) reweighting approach to interpret CCR rates of Ubiquitin, we demonstrate that these uncertainties do not necessarily impair resolving CCR-encoded structural information. Instead, a suitable balance between complementary CCR experiments and prior information is found to be the most crucial factor in mapping backbone dihedral angle distributions. Experimental and systematic deviations such as oversimplified dynamics appear to be of minor importance. Using Ubiquitin as an example, we demonstrate that CCR rates are capable of characterizing rigid and flexible residues alike, indicating their unharnessed potential in studying disordered proteins.

Association between Predicted Effects of TP53 Missense Variants on Protein Conformation and Their Phenotypic Presentation as Li-Fraumeni Syndrome or Hereditary Breast Cancer.

Rare germline pathogenic TP53 missense variants often predispose to a wide spectrum of tumors characterized by Li-Fraumeni syndrome (LFS) but a subset of variants is also seen in families with exclusively hereditary breast cancer (HBC) outcomes. We have developed a logistic regression model with the aim of predicting LFS and HBC outcomes, based on the predicted effects of individual TP53 variants on aspects of protein conformation. A total of 48 missense variants either unique for LFS (n = 24) or exclusively reported in HBC (n = 24) were included. LFS-variants were over-represented in residues tending to be buried in the core of the tertiary structure of TP53 (p = 0.0014). The favored logistic regression model describes disease outcome in terms of explanatory variables related to the surface or buried status of residues as well as their propensity to contribute to protein compactness or protein-protein interactions. Reduced, internally validated models discriminated well between LFS and HBC (C-statistic = 0.78-0.84; equivalent to the area under the ROC (receiver operating characteristic) curve), had a low risk for over-fitting and were well calibrated in relation to the known outcome risk. In conclusion, this study presents a phenotypic prediction model of LFS and HBC risk for germline TP53 missense variants, in an attempt to provide a complementary tool for future decision making and clinical handling.

A novel high-dimensional NMR experiment for resolving protein backbone dihedral angle ambiguities.

Intrinsically disordered proteins (IDPs) are challenging established structural biology perception and urge a reassessment of the conventional understanding of the subtle interplay between protein structure and dynamics. Due to their importance in eukaryotic life and central role in protein interaction networks, IDP research is a fascinating and highly relevant research area in which NMR spectroscopy is destined to be a key player. The flexible nature of IDPs, as a result of the sampling of a vast conformational space, however, poses a tremendous scientific challenge, both technically and theoretically. Pronounced signal averaging results in narrow signal dispersion and requires higher dimensionality NMR techniques. Moreover, a fundamental problem in the structural characterization of IDPs is the definition of the conformational ensemble sampled by the polypeptide chain in solution, where often the interpretation relies on the concept of 'residual structure' or 'conformational preference'. An important source of structural information is information-rich NMR experiments that probe protein backbone dihedral angles in a unique manner. Cross-correlated relaxation experiments have proven to fulfil this task as they provide unique information about protein backbones, particularly in IDPs. Here we present a novel cross-correlation experiment that utilizes non-uniform sampling detection schemes to resolve protein backbone dihedral ambiguities in IDPs. The sensitivity of this novel technique is illustrated with an application to the prototypical IDP [Formula: see text]-Synculein for which unexpected deviations from random-coil-like behaviour could be observed.

Sensitivity-enhanced three-dimensional and carbon-detected two-dimensional NMR of proteins using hyperpolarized water.

Signal enhancements of up to two orders of magnitude in protein NMR can be achieved by employing HDO as a vector to introduce hyperpolarization into folded or intrinsically disordered proteins. In this approach, hyperpolarized HDO produced by dissolution-dynamic nuclear polarization (D-DNP) is mixed with a protein solution waiting in a high-field NMR spectrometer, whereupon amide proton exchange and nuclear Overhauser effects (NOE) transfer hyperpolarization to the protein and enable acquisition of a signal-enhanced high-resolution spectrum. To date, the use of this strategy has been limited to 1D and 1H-15N 2D correlation experiments. Here we introduce 2D 13C-detected D-DNP, to reduce exchange-induced broadening and other relaxation penalties that can adversely affect proton-detected D-DNP experiments. We also introduce hyperpolarized 3D spectroscopy, opening the possibility of D-DNP studies of larger proteins and IDPs, where assignment and residue-specific investigation may be impeded by spectral crowding. The signal enhancements obtained depend in particular on the rates of chemical and magnetic exchange of the observed residues, thus resulting in non-uniform 'hyperpolarization-selective' signal enhancements. The resulting spectral sparsity, however, makes it possible to resolve and monitor individual amino acids in IDPs of over 200 residues at acquisition times of just over a minute. We apply the proposed experiments to two model systems: the compactly folded protein ubiquitin, and the intrinsically disordered protein (IDP) osteopontin (OPN).

19 F†NMR Spectroscopy Tagging and Paramagnetic Relaxation Enhancement-Based Conformation Analysis of Intrinsically Disordered Protein Complexes.

The combination of 19 F NMR spectroscopy tagging and paramagnetic relaxation enhancement (PRE) NMR spectroscopy experiments was evaluated as a versatile method to probe protein-protein interactions and conformational changes of intrinsically disordered proteins upon complex formation. The feasibility of the approach is illustrated with an application to the Myc-Max protein complex; this is an oncogenic transcription factor that binds enhancer box DNA fragments. The single cysteine residue of Myc was tagged with highly fluorinated [19 F]3,5-bis(trifluoromethyl)benzyl bromide. Structural dynamics of the protein complex were monitored through intermolecular PREs between 19 F-Myc and paramagnetic (1-oxyl-2,2,5,5-tetramethyl-Δ3-pyrroline-3-methyl)methanethiosulfonate (MTSL)-tagged) Max. The 19 F R2 relaxation rates obtained with three differently MTSL-tagged Max mutants revealed novel insights into the differential structural dynamics of Myc-Max bound to DNA and the tumour suppressor breast cancer antigen 1. Given its ease of implementation, fruitful applications of this strategy to structural biology and inhibitor screening can be envisaged.

NMR Characterization of Surface Receptor Protein Interactions in Live Cells Using Methylcellulose Hydrogels.

Interactions of transmembrane receptors with their extracellular ligands are essential for cellular communication and signaling and are therefore a major focus in drug discovery programs. The transition from in vitro to live cell interaction studies, however, is typically a bottleneck in many drug discovery projects due to the challenge of obtaining atomic-resolution information under near-physiological conditions. Although NMR spectroscopy is ideally suited to overcome this limitation, several experimental impairments are still present. Herein, we propose the use of methylcellulose hydrogels to study extracellular proteins and their interactions with plasma membrane receptors. This approach reduces cell sedimentation, prevents the internalization of membrane receptors, and increases cell survival, while retaining the free tumbling of extracellular proteins.

PI by NMR: Probing CH-π Interactions in Protein-Ligand Complexes by NMR Spectroscopy.

While CH-π interactions with target proteins are crucial determinants for the affinity of arguably every drug molecule, no method exists to directly measure the strength of individual CH-π interactions in drug-protein complexes. Herein, we present a fast and reliable methodology called PI (π interactions) by NMR, which can differentiate the strength of protein-ligand CH-π interactions in solution. By combining selective amino-acid side-chain labeling with 1 H-13 C NMR, we are able to identify specific protein protons of side-chains engaged in CH-π interactions with aromatic ring systems of a ligand, based solely on 1 H chemical-shift values of the interacting protein aromatic ring protons. The information encoded in the chemical shifts induced by such interactions serves as a proxy for the strength of each individual CH-π interaction. PI by NMR changes the paradigm by which chemists can optimize the potency of drug candidates: direct determination of individual π interactions rather than averaged measures of all interactions.

NMR Characterization of Long-Range Contacts in Intrinsically Disordered Proteins from Paramagnetic Relaxation Enhancement in 13 C Direct-Detection Experiments.

Intrinsically disordered proteins (IDPs) carry out many biological functions. They lack a stable 3D structure and are able to adopt many different conformations in dynamic equilibrium. The interplay between local dynamics and global rearrangements is key for their function. A widely used experimental NMR spectroscopy approach to study long-range contacts in IDPs exploits paramagnetic effects, and 1 H detection experiments are generally used to determine paramagnetic relaxation enhancement (PRE) for amide protons. However, under physiological conditions, exchange broadening hampers the detection of solvent-exposed amide protons, which reduces the content of information available. Herein, we present an experimental approach based on direct carbon detection of PRE that provides improved resolution, reduced sensitivity to exchange broadening, and complementary information derived from the use of different starting polarization sources.