A set of cross-correlated relaxation experiments to probe the correlation time of two different and complementary spin pairs.

Intrinsically disordered proteins (IDPs) defy the conventional structure-function paradigm by lacking a well-defined tertiary structure and exhibiting inherent flexibility. This flexibility leads to distinctive spin relaxation modes, reflecting isolated and specific motions within individual peptide planes. In this work, we propose a new pulse sequence to measure the longitudinal 13C' CSA-13C'-13Cα DD CCR rate [Formula: see text] and present a novel 3D version of the transverse [Formula: see text] CCR rate, adopting the symmetrical reconversion approach. We combined these rates with the analogous ΓxyN/NH and ΓzN/NH CCR rates to derive residue-specific correlation times for both spin-pairs within the same peptide plane. The presented approach offers a straightforward and intuitive way to compare the correlation times of two different and complementary spin vectors, anticipated to be a valuable aid to determine IDPs backbone dihedral angles distributions. We performed the proposed experiments on two systems: a folded protein ubiquitin and Coturnix japonica osteopontin, a prototypical IDP. Comparative analyses of the results show that the correlation times of different residues vary more for IDPs than globular proteins, indicating that the dynamics of IDPs is largely heterogeneous and dominated by local fluctuations.

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.

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.

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.

Optimal 13C NMR investigation of intrinsically disordered proteins at 1.2 GHz.

Nuclear magnetic resonance (NMR) spectroscopy is a powerful technique for characterizing biomolecules such as proteins and nucleic acids at atomic resolution. Increased magnetic field strengths drive progress in biomolecular NMR applications, leading to improved performance, e.g., higher resolution. A new class of NMR spectrometers with a 28.2 T magnetic field (1.2 GHz 1H frequency) has been commercially available since the end of 2019. The availability of ultra-high-field NMR instrumentation makes it possible to investigate more complex systems using NMR. This is especially true for highly flexible intrinsically disordered proteins (IDPs) and highly flexible regions (IDRs) of complex multidomain proteins. Indeed, the investigation of these proteins is frequently hampered by the crowding of NMR spectra. The advantages, however, are accompanied by challenges that the user must overcome when conducting experiments at such a high field (e.g., large spectral widths, radio frequency bandwidth, performance of decoupling schemes). This protocol presents strategies and tricks for optimising high-field NMR experiments for IDPs/IDRs based on the analysis of the relaxation properties of the investigated protein. The protocol, tested on three IDPs of different molecular weight and structural complexity, focuses on 13C-detected NMR at 1.2 GHz. A set of experiments, including some multiple receiver experiments, and tips to implement versions tailored for IDPs/IDRs are described. However, the general approach and most considerations can also be applied to experiments that acquire 1H or 15N nuclei and to experiments performed at lower field strengths.

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.

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.

Harnessing PROTAC technology to combat stress hormone receptor activation.

Counteracting the overactivation of glucocorticoid receptors (GR) is an important therapeutic goal in stress-related psychiatry and beyond. The only clinically approved GR antagonist lacks selectivity and induces unwanted side effects. To complement existing tools of small-molecule-based inhibitors, we present a highly potent, catalytically-driven GR degrader, KH-103, based on proteolysis-targeting chimera technology. This selective degrader enables immediate and reversible GR depletion that is independent of genetic manipulation and circumvents transcriptional adaptations to inhibition. KH-103 achieves passive inhibition, preventing agonistic induction of gene expression, and significantly averts the GR's genomic effects compared to two currently available inhibitors. Application in primary-neuron cultures revealed the dependency of a glucocorticoid-induced increase in spontaneous calcium activity on GR. Finally, we present a proof of concept for application in vivo. KH-103 opens opportunities for a more lucid interpretation of GR functions with translational potential.

Studies of proline conformational dynamics in IDPs by 13C-detected cross-correlated NMR relaxation.

Intrinsically disordered proteins (IDPs) are significantly enriched in proline residues, which can populate specific local secondary structural elements called PPII helices, characterized by small packing densities. Proline is often thought to promote disorder, but it can participate in specific π·CH interactions with aromatic side chains resulting in reduced conformational flexibilities of the polypeptide. Differential local motional dynamics are relevant for the stabilization of preformed structural elements and can serve as nucleation sites for the establishment of long-range interactions. NMR experiments to probe the dynamics of proline ring systems would thus be highly desirable. Here we present a pulse scheme based on 13C detection to quantify dipole-dipole cross-correlated relaxation (CCR) rates at methylene CH2 groups in proline residues. Applying 13C-CON detection strategy provides exquisite spectral resolution allowing applications also to high molecular weight IDPs even in conditions approaching the physiological ones. The pulse scheme is illustrated with an application to the 220 amino acids long protein Osteopontin, an extracellular cytokine involved in inflammation and cancer progression, and a construct in which three proline-aromatic sequence patches have been mutated.

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.

Crucial neuroprotective roles of the metabolite BH4 in dopaminergic neurons.

Dopa-responsive dystonia (DRD) and Parkinson's disease (PD) are movement disorders caused by the dysfunction of nigrostriatal dopaminergic neurons. Identifying druggable pathways and biomarkers for guiding therapies is crucial due to the debilitating nature of these disorders. Recent genetic studies have identified variants of GTP cyclohydrolase-1 (GCH1), the rate-limiting enzyme in tetrahydrobiopterin (BH4) synthesis, as causative for these movement disorders. Here, we show that genetic and pharmacological inhibition of BH4 synthesis in mice and human midbrain-like organoids accurately recapitulates motor, behavioral and biochemical characteristics of these human diseases, with severity of the phenotype correlating with extent of BH4 deficiency. We also show that BH4 deficiency increases sensitivities to several PD-related stressors in mice and PD human cells, resulting in worse behavioral and physiological outcomes. Conversely, genetic and pharmacological augmentation of BH4 protects mice from genetically- and chemically induced PD-related stressors. Importantly, increasing BH4 levels also protects primary cells from PD-affected individuals and human midbrain-like organoids (hMLOs) from these stressors. Mechanistically, BH4 not only serves as an essential cofactor for dopamine synthesis, but also independently regulates tyrosine hydroxylase levels, protects against ferroptosis, scavenges mitochondrial ROS, maintains neuronal excitability and promotes mitochondrial ATP production, thereby enhancing mitochondrial fitness and cellular respiration in multiple preclinical PD animal models, human dopaminergic midbrain-like organoids and primary cells from PD-affected individuals. Our findings pinpoint the BH4 pathway as a key metabolic program at the intersection of multiple protective mechanisms for the health and function of midbrain dopaminergic neurons, identifying it as a potential therapeutic target for PD.

Early intervention with azelastine nasal spray may reduce viral load in SARS-CoV-2 infected patients.

With the changing epidemiology of COVID-19 and its impact on our daily lives, there is still an unmet need of COVID-19 therapies treating early infections to prevent progression. The current study was a randomized, parallel, double-blind, placebo-controlled trial. Ninety SARS-CoV-2 positive patients were randomized into 3 groups receiving placebo, 0.02% or 0.1% azelastine nasal spray for 11 days, during which viral loads were assessed by quantitative PCR. Investigators assessed patients' status throughout the trial including safety follow-ups (days 16 and 60). Symptoms were documented in patient diaries. Initial viral loads were log10 6.85 ± 1.31 (mean ± SD) copies/mL (ORF 1a/b gene). After treatment, virus load was reduced in all groups (p < 0.0001) but was greater in the 0.1% group compared to placebo (p = 0.007). In a subset of patients (initial Ct < 25) viral load was strongly reduced on day 4 in the 0.1% group compared to placebo (p = 0.005). Negative PCR results appeared earlier and more frequently in the azelastine treated groups: being 18.52% and 21.43% in the 0.1% and 0.02% groups, respectively, compared to 0% for placebo on day 8. Comparable numbers of adverse events occurred in all treatment groups with no safety concerns. The shown effects of azelastine nasal spray may thus be suggestive of azelastine's potential as an antiviral treatment.Trial registration: The study was registered in the German Clinical Trial Register (DRKS-ID: DRKS00024520; Date of Registration in DRKS: 12/02/2021). EudraCT number: 2020-005544-34.

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.

The Anti-Histamine Azelastine, Identified by Computational Drug Repurposing, Inhibits Infection by Major Variants of SARS-CoV-2 in Cell Cultures and Reconstituted Human Nasal Tissue.

Background and purpose: The COVID-19 pandemic continues to pose challenges, especially with the emergence of new SARS-CoV-2 variants that are associated with higher infectivity and/or compromised protection afforded by the current vaccines. There is a high demand for additional preventive and therapeutic strategies effective against this changing virus. Repurposing of approved or clinically tested drugs can provide an immediate solution. Experimental Approach: We applied a novel computational approach to search among approved and commercially available drugs. Antiviral activity of a predicted drug, azelastine, was tested in vitro in SARS-CoV-2 infection assays with Vero E6 cells, Vero cells stably overexpressing the human TMPRSS2 and ACE2 proteins as well as on reconstituted human nasal tissue using the predominant variant circulating in Europe in summer 2020, B.1.177 (D614G variant), and its emerging variants of concern; B.1.1.7 (alpha), B.1.351 (beta) and B.1.617.2 (delta) variants. The effect of azelastine on viral replication was assessed by quantification of viral genomes by droplet digital PCR or qPCR. Key results: The computational approach identified major drug families, such as anti-infective, anti-inflammatory, anti-hypertensive, antihistamine, and neuroactive drugs. Based on its attractive safety profile and availability in nasal formulation, azelastine, a histamine 1 receptor-blocker was selected for experimental testing. Azelastine reduced the virus-induced cytopathic effect and SARS-CoV-2 copy numbers both in preventive and treatment settings upon infection of Vero cells with an EC50 of 2.2-6.5 µM. Comparable potency was observed with the alpha, beta and delta variants. Furthermore, five-fold dilution (containing 0.02% azelastine) of the commercially available nasal spray formulation was highly potent in inhibiting viral propagation in reconstituted human nasal tissue. Conclusion and Implications: Azelastine, an antihistamine available as nasal sprays developed against allergic rhinitis may be considered as a topical prevention or treatment of nasal colonization by SARS-CoV-2. A Phase 2 efficacy indicator study with azelastine-containing nasal spray that was designed based on the findings reported here has been concluded recently, confirming accelerated viral clearance in SARS-CoV-2 positive subjects.

Long-range structural preformation in yes-associated protein precedes encounter complex formation with TEAD.

Yes-associated protein (YAP) is a partly intrinsically disordered protein (IDP) that plays a major role as the downstream element of the Hippo pathway. Although the structures of the complex between TEA domain transcription factors (TEADs) and the TEAD-binding domain of YAP are already well characterized, its apo state and the binding mechanism with TEADs are still not clearly defined. Here we characterize via a combination of different NMR approaches with site-directed mutagenesis and affinity measurements the intrinsically disordered solution state of apo YAP. Our results provide evidence that the apo state of YAP adopts several compact conformations that may facilitate the formation of the YAP:TEAD complex. The interplay between local secondary structure element preformation and long-range co-stabilization of these structured elements precedes the encounter complex formation with TEAD and we, therefore, propose that TEAD binding proceeds largely via conformational selection of the preformed compact substates displaying at least nanosecond lifetimes.

A Step toward NRF2-DNA Interaction Inhibitors by Fragment-Based NMR Methods.

The NRF2 transcription factor is a key regulator in cellular oxidative stress response, and acts as a tumor suppressor. Aberrant activation of NRF2 has been implicated in promoting chemo-resistance, tumor growth, and metastasis by activating its downstream target genes. Hence, inhibition of NRF2 promises to be an attractive therapeutic strategy to suppress cell proliferation and enhance cell apoptosis in cancer. Direct targeting of NRF2 with small-molecules to discover protein-DNA interaction inhibitors is challenging as it is a largely intrinsically disordered protein. To discover molecules that bind to NRF2 at the DNA binding interface, we performed an NMR-based fragment screen against its DNA-binding domain. We discovered several weakly binding fragment hits that bind to a region overlapping with the DNA binding site. Using SAR by catalogue we developed an initial structure-activity relationship for the most interesting initial hit series. By combining NMR chemical shift perturbations and data-driven docking, binding poses which agreed with NMR information and the observed SAR were elucidated. The herein discovered NRF2 hits and proposed binding modes form the basis for future structure-based optimization campaigns on this important but to date 'undrugged' cancer driver.

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.

On-Cell NMR Contributions to Membrane Receptor Binding Characterization.

NMR spectroscopy has matured into a powerful tool to characterize interactions between biological molecules at atomic resolution, most importantly even under near to native (physiological) conditions. The field of in-cell NMR aims to study proteins and nucleic acids inside living cells. However, cells interrogate their environment and are continuously modulated by external stimuli. Cell signaling processes are often initialized by membrane receptors on the cell surface; therefore, characterizing their interactions at atomic resolution by NMR, hereafter referred as on-cell NMR, can provide valuable mechanistic information. This review aims to summarize recent on-cell NMR tools that give information about the binding site and the affinity of membrane receptors to their ligands together with potential applications to in vivo drug screening systems.

Order from disorder in the sarcomere: FATZ forms a fuzzy but tight complex and phase-separated condensates with α-actinin.

In sarcomeres, α-actinin cross-links actin filaments and anchors them to the Z-disk. FATZ (filamin-, α-actinin-, and telethonin-binding protein of the Z-disk) proteins interact with α-actinin and other core Z-disk proteins, contributing to myofibril assembly and maintenance. Here, we report the first structure and its cellular validation of α-actinin-2 in complex with a Z-disk partner, FATZ-1, which is best described as a conformational ensemble. We show that FATZ-1 forms a tight fuzzy complex with α-actinin-2 and propose an interaction mechanism via main molecular recognition elements and secondary binding sites. The obtained integrative model reveals a polar architecture of the complex which, in combination with FATZ-1 multivalent scaffold function, might organize interaction partners and stabilize α-actinin-2 preferential orientation in Z-disk. Last, we uncover FATZ-1 ability to phase-separate and form biomolecular condensates with α-actinin-2, raising the question whether FATZ proteins can create an interaction hub for Z-disk proteins through membraneless compartmentalization during myofibrillogenesis.

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.