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.

A Switch between Two Intrinsically Disordered Conformational Ensembles Modulates the Active Site of a Basic-Helix-Loop-Helix Transcription Factor.

We report a conformational switch between two distinct intrinsically disordered subensembles within the active site of a transcription factor. This switch highlights an evolutionary benefit conferred by the high plasticity of intrinsically disordered domains, namely, their potential to dynamically sample a heterogeneous conformational space housing multiple states with tailored properties. We focus on proto-oncogenic basic-helix-loop-helix (bHLH)-type transcription factors, as these play key roles in cell regulation and function. Despite intense research efforts, the understanding of structure-function relations of these transcription factors remains incomplete as they feature intrinsically disordered DNA-interaction domains that are difficult to characterize, theoretically as well as experimentally. Here we characterize the structural dynamics of the intrinsically disordered region DNA-binding site of the vital MYC-associated transcription factor X (MAX). Integrating nuclear magnetic resonance (NMR) measurements, molecular dynamics (MD) simulations, and electron paramagnetic resonance (EPR) measurements, we show that, in the absence of DNA, the binding site of the free MAX2 homodimer samples two intrinsically disordered conformational subensembles. These feature distinct structural properties: one subensemble consists of a set of highly flexible and spatially extended conformers, while the second features a set of "hinged" conformations. In this latter ensemble, the disordered N-terminal tails of MAX2 fold back along the dimer, forming transient long-range contacts with the HLH-region and thereby exposing the DNA binding site to the solvent. The features of these divergent substates suggest two mechanisms by which protein conformational dynamics in MAX2 might modulate DNA-complex formation: by enhanced initial recruitment of free DNA ligands, as a result of the wider conformational space sampled by the extended ensemble, and by direct exposure of the binding site and the corresponding strong electrostatic attractions presented while in the hinged conformations.

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.

Author Correction: Binding of the protein ICln to α-integrin contributes to the activation of IClswell current.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Binding of the protein ICln to α-integrin contributes to the activation of IClswell current.

IClswell is the chloride current induced by cell swelling, and plays a fundamental role in several biological processes, including the regulatory volume decrease (RVD). ICln is a highly conserved, ubiquitously expressed and multifunctional protein involved in the activation of IClswell. In platelets, ICln binds to the intracellular domain of the integrin αIIb chain, however, whether the ICln/integrin interaction plays a role in RVD is not known. Here we show that a direct molecular interaction between ICln and the integrin α-chain is not restricted to platelets and involves highly conserved amino acid motifs. Integrin α recruits ICln to the plasma membrane, thereby facilitating the activation of IClswell during hypotonicity. Perturbation of the ICln/integrin interaction prevents the transposition of ICln towards the cell surface and, in parallel, impedes the activation of IClswell. We suggest that the ICln/integrin interaction interface may represent a new molecular target enabling specific IClswell suppression in pathological conditions when this current is deregulated or plays a detrimental role.

Conformational tuning of a DNA-bound transcription factor.

Transcription factors are involved in many cellular processes that take place remote from their cognate DNA sequences. The efficiencies of these activities are thus in principle counteracted by high binding affinities of the factors to their cognate DNAs. Models such as facilitated diffusion or dissociation address this apparent contradiction. We show that the MYC associated transcription factor X (MAX) undergoes nanoscale conformational fluctuations in the DNA-bound state, which is consistent with facilitated dissociation from or diffusion along DNA strands by transiently reducing binding energies. An integrative approach involving EPR, NMR, crystallographic and molecular dynamics analyses demonstrates that the N-terminal domain of MAX constantly opens and closes around a bound DNA ligand thereby dynamically tuning the binding epitope and the mode of interaction.

Hydrophobic Collapse of the Intrinsically Disordered Transcription Factor Myc Associated Factor X.

The conformational space of the proto-oncogenic transcription factor Myc associated factor X (MAX) comprises a dynamic equilibrium between a stably folded coiled-coil homodimer and an intrinsically disordered ensemble of states. We show by means of nuclear magnetic resonance spectroscopy that the intrinsically disordered ensemble samples structures that are even as compact as the folded dimer. These extremely dense, hydrophobically collapsed globules might be of importance for interconversion between different conformations of intrinsically disordered proteins.

Calcium-dependent binding of Myc to calmodulin.

The bHLH-LZ (basic region/helix-loop-helix/leucine zipper) oncoprotein Myc and the bHLH-LZ protein Max form a binary transcription factor complex controlling fundamental cellular processes. Deregulated Myc expression leads to neoplastic transformation and is a hallmark of most human cancers. The dynamics of Myc transcription factor activity are post-translationally coordinated by defined protein-protein interactions. Here, we present evidence for a second messenger controlled physical interaction between the Ca2+ sensor calmodulin (CaM) and all Myc variants (v-Myc, c-Myc, N-Myc, and L-Myc). The predominantly cytoplasmic Myc:CaM interaction is Ca2+-dependent, and the binding site maps to the conserved bHLH domain of Myc. Ca2+-loaded CaM binds the monomeric and intrinsically disordered Myc protein with high affinity, whereas Myc:Max heterodimers show less, and Max homodimers no affinity for CaM. NMR spectroscopic analyses using alternating mixtures of 15N-labeled and unlabeled preparations of CaM and a monomeric Myc fragment containing the bHLH-LZ domain corroborate the biochemical results on the Myc:CaM interaction and confirm the interaction site mapping. In electrophoretic mobility shift assays, addition of CaM does not affect high-affinity DNA-binding of Myc:Max heterodimers. However, cell-based reporter analyses and cell transformation assays suggest that increasing CaM levels enhance Myc transcriptional and oncogenic activities. Our results point to a possible involvement of Ca2+ sensing CaM in the fine-tuning of Myc function.

A de novo compound targeting α-synuclein improves deficits in models of Parkinson's disease.

Abnormal accumulation and propagation of the neuronal protein α-synuclein has been hypothesized to underlie the pathogenesis of Parkinson's disease, dementia with Lewy bodies and multiple system atrophy. Here we report a de novo-developed compound (NPT100-18A) that reduces α-synuclein toxicity through a novel mechanism that involves displacing α-synuclein from the membrane. This compound interacts with a domain in the C-terminus of α-synuclein. The E83R mutation reduces the compound interaction with the 80-90 amino acid region of α-synuclein and prevents the effects of NPT100-18A. In vitro studies showed that NPT100-18A reduced the formation of wild-type α-synuclein oligomers in membranes, reduced the neuronal accumulation of α-synuclein, and decreased markers of cell toxicity. In vivo studies were conducted in three different α-synuclein transgenic rodent models. Treatment with NPT100-18A ameliorated motor deficits in mThy1 wild-type α-synuclein transgenic mice in a dose-dependent manner at two independent institutions. Neuropathological examination showed that NPT100-18A decreased the accumulation of proteinase K-resistant α-synuclein aggregates in the CNS and was accompanied by the normalization of neuronal and inflammatory markers. These results were confirmed in a mutant line of α-synuclein transgenic mice that is prone to generate oligomers. In vivo imaging studies of α-synuclein-GFP transgenic mice using two-photon microscopy showed that NPT100-18A reduced the cortical synaptic accumulation of α-synuclein within 1 h post-administration. Taken together, these studies support the notion that altering the interaction of α-synuclein with the membrane might be a feasible therapeutic approach for developing new disease-modifying treatments of Parkinson's disease and other synucleinopathies.

Five and four dimensional experiments for robust backbone resonance assignment of large intrinsically disordered proteins: application to Tau3x protein.

New experiments dedicated for large IDPs backbone resonance assignment are presented. The most distinctive feature of all described techniques is the employment of MOCCA-XY16 mixing sequences to obtain effective magnetization transfers between carbonyl carbon backbone nuclei. The proposed 4 and 5 dimensional experiments provide a high dispersion of obtained signals making them suitable for use in the case of large IDPs (application to 354 a. a. residues of Tau protein 3x isoform is presented) as well as provide both forward and backward connectivities. What is more, connecting short chains interrupted with proline residues is also possible. All the experiments employ non-uniform sampling.

Direct methods and residue type specific isotope labeling in NMR structure determination and model-driven sequential assignment.

Direct methods in NMR based structure determination start from an unassigned ensemble of unconnected gaseous hydrogen atoms. Under favorable conditions they can produce low resolution structures of proteins. Usually a prohibitively large number of NOEs is required, to solve a protein structure ab-initio, but even with a much smaller set of distance restraints low resolution models can be obtained which resemble a protein fold. One problem is that at such low resolution and in the absence of a force field it is impossible to distinguish the correct protein fold from its mirror image. In a hybrid approach these ambiguous models have the potential to aid in the process of sequential backbone chemical shift assignment when (13)C(beta) and (13)C' shifts are not available for sensitivity reasons. Regardless of the overall fold they enhance the information content of the NOE spectra. These, combined with residue specific labeling and minimal triple-resonance data using (13)C(alpha) connectivity can provide almost complete sequential assignment. Strategies for residue type specific labeling with customized isotope labeling patterns are of great advantage in this context. Furthermore, this approach is to some extent error-tolerant with respect to data incompleteness, limited precision of the peak picking, and structural errors caused by misassignment of NOEs.

Generation and relaxation of high rank coherences in AX3 systems in a selectively methionine labelled SH2 domain.

The usefulness of selective isotope labelling patterns is demonstrated using the C-terminal SH2 domain of PLC-gamma1 selectively 13C labelled at methionine methyl groups. We demonstrate the generation and relaxation of coherences that are second rank in protons and first rank in carbons that derive from quadrupolar order in protons. The decay rates of second rank double quantum proton coherences are measured. These terms exhibit fewer channels for cross-correlated relaxation compared to single quantum coherences. Our results indicate the potential application of the measurement of high order proton coherences to the analysis of dynamics in methyl-bearing side chains.