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An unambiguous description of an experimental setup and analysis, and the subsequent biological observation is vital for accurate data interpretation and reproducible results. Consequently, experimental analyses should be described in a concise, unequivocal, and digestible manner. The aim of minimum information guidelines is to define the fundamental complement of data that can support an unambiguous conclusion on experimental observations. In this document, we present the Minimum Information About Disorder Experiments (MIADE) guidelines to define the minimal fundamental parameters required for non-experts to understand the key findings of an experiment studying intrinsically disordered proteins (IDPs) or intrinsically disordered protein regions (IDRs). MIADE guidelines provide recommendations for data producers to describe the results of their experiments at source, for curators to annotate experimental data to community resources and for database developers maintaining community resources to disseminate the data. We give examples of the application of these guidelines in common use cases and describe the implementation of an update to the DisProt IDP database to allow MIADE-compliant annotation. The MIADE guidelines will improve the interpretability of experimental results for data consumers, facilitate direct data submission, simplify data curation, improve data exchange among repositories and standardise the dissemination of the key metadata on an IDP experiment by IDP data sources.

International audience; Copper/zinc superoxide dismutase (SOD) is a homodimeric metalloenzyme which has been extensively studied as a benchmark for structure-function relationships in proteins, in particular because of its implication in the familial form of the neurodegenerative disease amyotrophic lateral sclerosis. Here, we investigate microcrystalline preparations of two differently metallated forms of SOD, namely the fully mature functional Cu I ,Zn state and the E,Zn-SOD state in which the Cu site is empty. By using solid-state NMR with fast magic-angle spinning (MAS) at high magnetic fields (1 H Larmor frequency of 800-1000 MHz), we quantify motions spanning a dynamic range from ns to ms. We determine that metal ion uptake does not act as a rigidification element but as a switch redistributing motional processes on different timescales, with coupling of the dynamics of histidine sidechains and those of remote key backbone elements of the protein.

Many properties of intrinsically disordered proteins (IDPs), or protein regions (IDRs), are modulated by the nature of amino acid side chains as well as by local solvent exposure. We propose a set of exclusively heteronuclear NMR experiments to investigate these features in different experimental conditions that are relevant for physiological function. The proposed approach is generally applicable to many IDPs/IDRs whose assignment is available in the Biological Magnetic Resonance Bank (BMRB) to investigate how their properties are modulated by different, physiologically relevant conditions. The experiments, tested on α-synuclein, are then used to investigate how α-synuclein senses Ca2+ concentration jumps associated with the transmission of nerve signals. Novel modules in the primary sequence of α-synuclein optimized for calcium sensing in highly flexible, disordered protein segments are identified.

Intrinsically disordered proteins (IDPs) carry out many biological functions. They lack a stable three-dimensional structure, but rather adopt many different conformations in dynamic equilibrium. The interplay between local dynamics and global rearrangements is key for their function. In IDPs, proline residues are significantly enriched. Given their unique physicochemical and structural properties, a more detailed understanding of their potential role in stabilizing partially folded states in IDPs is highly desirable. Nuclear magnetic resonance (NMR) spectroscopy, and in particular 13C-detected NMR, is especially suitable to address these questions. We applied a 13C-detected strategy to study Osteopontin, a largely disordered IDP with a central compact region. By using the exquisite sensitivity and spectral resolution of these novel techniques, we gained unprecedented insight into cis-Pro populations, their local structural dynamics, and their role in mediating long-range contacts. Our findings clearly call for a reassessment of the structural and functional role of proline residues in IDPs. The emerging picture shows that proline residues have ambivalent structural roles. They are not simply disorder promoters but rather can, depending on the primary sequence context, act as nucleation sites for structural compaction in IDPs. These unexpected features provide a versatile mechanistic toolbox to enrich the conformational ensembles of IDPs with specific features for adapting to changing molecular and cellular environments.

The knowledge of interactions between different molecules is undoubtedly the driving force of all contemporary biomedical and biological sciences. Chemical biology/biological chemistry has become an important multidisciplinary bridge connecting the perspectives of chemistry and biology to the study of small molecules/peptidomimetics and their interactions in biological systems. Advances in structural biology research, in particular linking atomic structure to molecular properties and cellular context, are essential for the sophisticated design of new medicines that exhibit a high degree of druggability and very importantly, druglikeness. The authors of this contribution are outstanding scientists in the field who provided a brief overview of their work, which is arranged from in silico investigation through the characterization of interactions of compounds with biomolecules to bioactive materials.

International audience; Henipaviruses are BSL-4 zoonotic pathogens responsible in humans for severe encephalitis. Their V protein is a key player in the evasion of the host innate immune response. We previously showed that the Henipavirus V proteins consist of a long intrinsically disordered N-terminal domain (NTD) and a β-enriched C-terminal domain (CTD). These terminals are critical for V binding to DDB1, which is a cellular protein that is a component of the ubiquitin ligase E3 complex, as well as binding to MDA5 and LGP2, which are two host sensors of viral RNA. Here, we serendipitously discovered that the Hendra virus V protein undergoes a liquid-to-hydrogel phase transition and identified the V region responsible for this phenomenon. This region, referred to as PNT3 and encompassing residues 200–310, was further investigated using a combination of biophysical and structural approaches. Congo red binding assays, together with negative-staining transmisison electron microscopy (TEM) studies, show that PNT3 forms amyloid-like fibrils. Fibrillation abilities are dramatically reduced in a rationally designed PNT3 variant in which a stretch of three contiguous tyrosines, falling within an amyloidogenic motif, were replaced by three alanines. Worthy to note, Congo red staining experiments provided hints that these amyloid-like fibrils form not only in vitro but also in cellula after transfection or infection. The present results set the stage for further investigations aimed at assessing the functional role of phase separation and fibrillation by the Henipavirus V proteins.

When present, structural disorder makes it very challenging to characterise the conformational properties of proteins. This is particularly the case of proteins, such as the oncogene protein E7 of human papillomavirus type 16, which contain both ordered and disordered domains, and that can populate monomeric and oligomeric states under physiological conditions. Nuclear magnetic resonance (NMR) spectroscopy is emerging as a powerful method to study these complex systems, most notably in combination with molecular dynamics simulations. Here we use NMR chemical shifts and residual dipolar couplings as structural restraints in replica-averaged molecular dynamics simulations to determine the free energy landscape of E7. This landscape reveals a complex interplay between a folded but highly dynamical C-terminal domain and a disordered N-terminal domain that forms transient secondary and tertiary structures, as well as an equilibrium between a high-populated (98%) dimeric state and a low-populated (2%) monomeric state. These results provide compelling evidence of the complex conformational heterogeneity associated with the behaviour and interactions of this disordered protein associated with disease.

14 pags., 5 figs., 1 tab., The intracellular environment is crowded with macromolecules, including sugars, proteins and nucleic acids. In the cytoplasm, crowding effects are capable of excluding up to 40% of the volume available to any macromolecule when compared to dilute conditions. NUPR1 is an intrinsically disordered protein (IDP) involved in cell-cycle regulation, stress-cell response, apoptosis processes, DNA binding and repair, chromatin remodeling and transcription. Simulations of molecular crowding predict that IDPs can adopt compact states, as well as more extended conformations under crowding conditions. In this work, we analyzed the conformation and dynamics of NUPR1 in the presence of two synthetic polymers, Ficoll-70 and Dextran-40, which mimic crowding effects in the cells, at two different concentrations (50 and 150 mg/ml). The study was carried out by using a multi-spectroscopic approach, including: site-directed spin labelling electron paramagnetic resonance spectroscopy (SDSL-EPR), nuclear magnetic resonance spectroscopy (NMR), circular dichroism (CD), small angle X-ray scattering (SAXS) and dynamic light scattering (DLS). SDSL-EPR spectra of two spin-labelled mutants indicate that there was binding with the crowders and that the local dynamics of the C and N termini of NUPR1 were partially affected by the crowders. However, the overall disordered nature of NUPR1 did not change substantially in the presence of the crowders, as shown by circular dichroism CD and NMR, and further confirmed by EPR. The changes in the dynamics of the paramagnetic probes appear to be related to preferred local conformations and thus crowding agents partially affect some specific regions, further pinpointing that NUPR1 flexibility has a key physiological role in its activity., This work was supported by Spanish Ministry of Economy and Competitiveness and European ERDF Funds (MCIU/AEI/ FEDER, EU) (RTI 2018-097991-B-I00 to JN and PGC 2018- 094548-B-I00 to AA and PM), and the Basque Government (IT1175-19 to AA and PM).

NMR represents a key spectroscopic technique that contributes to the emerging field of highly flexible, intrinsically disordered proteins (IDPs) or protein regions (IDRs) that lack a stable three-dimensional structure. A set of exclusively heteronuclear NMR experiments tailored for proline residues, highly abundant in IDPs/IDRs, are presented here. They provide a valuable complement to the widely used approach based on amide proton detection, filling the gap introduced by the lack of amide protons in proline residues within polypeptide chains. The novel experiments have very interesting properties for the investigations of IDPs/IDRs of increasing complexity.

Direct interaction between intrinsically disordered proteins (IDPs) is often difficult to characterize hampering the elucidation of their binding mechanism. Particularly challenging is the study of fuzzy complexes, in which the intrinsically disordered proteins or regions retain conformational freedom within the assembly. To date, nuclear magnetic resonance spectroscopy has proven to be one of the most powerful techniques to characterize at the atomic level intrinsically disordered proteins and their interactions, including those cases where the formed complexes are highly dynamic. Here, we present the characterization of the interaction between a viral protein, the Early region 1A protein from Adenovirus (E1A), and a disordered region of the human CREB-binding protein, namely the fourth intrinsically disordered linker CBP-ID4. E1A was widely studied as a prototypical viral oncogene. Its interaction with two folded domains of CBP was mapped, providing hints for understanding some functional aspects of the interaction with this transcriptional coactivator. However, the role of the flexible linker connecting these two globular domains of CBP in this interaction was never explored before.

Most of our understanding of chemistry derives from atomic-level structures obtained with single-crystal X-ray diffraction. Metal centers in X-ray structures of small organometallic or coordination complexes are often extremely well-defined, with errors in the positions on the order of 10(-4)-10(-5) A. Determining the metal coordination geometry to high accuracy is essential for understanding metal center reactivity, as even small structural changes can dramatically alter the metal activity. In contrast, the resolution of X-ray structures in proteins is limited typically to the order of 10(-1) angstrom. This resolution is often not sufficient to develop precise structure-activity relations for the metal sites in proteins, because the uncertainty in positions can cover all of the known ranges of bond lengths and bond angles for a given type of metal complex. Here we introduce a new approach that enables the determination of a high-definition structure of the active site of a metalloprotein from a powder sample, by combining magic-angle spinning (MAS) nuclear magnetic resonance (NMR) spectroscopy, tailored radio frequency (RF) irradiation schemes, and computational approaches. This allows us to overcome the "blind sphere" in paramagnetic proteins, and to observe and assign H-1, C-13, and N-15 resonances for the ligands directly coordinating the metal center. We illustrate the method by determining the bond lengths in the structure of the Co-II coordination sphere at the core of human superoxide dismutase 1 (SOD) with 0.7 pm precision. The coordination geometry of the resulting structure explains the nonreactive nature of the Co-II/Zn-II centers in these proteins, which allows them to play a purely structural role.

Structural disorder represents a key feature in the mechanism of action of RNA-binding proteins (RBPs). Recent insights revealed that intrinsically disordered regions (IDRs) linking globular domains modulate their capability to interact with various sequences of RNA, but also regulate aggregation processes, stress-granules formation, and binding to other proteins. The FET protein family, which includes FUS (Fused in Sarcoma), EWG (Ewing Sarcoma) and TAF15 (TATA binding association factor 15) proteins, is a group of RBPs containing three different long IDRs characterized by the presence of RGG motifs. In this study, we present the characterization of a fragment of FUS comprising two RGG regions flanking the RNA Recognition Motif (RRM) alone and in the presence of a stem-loop RNA. From a combination of EPR and NMR spectroscopies, we established that the two RGG regions transiently interact with the RRM itself. These interactions may play a role in the recognition of stem-loop RNA, without a disorder-to-order transition but retaining high dynamics.

Many properties of intrinsically disordered proteins (IDPs), or protein regions (IDRs), are modulated by the nature of amino acid side chains as well as by local solvent exposure. We propose a set of exclusively heteronuclear NMR experiments to investigate these features in different experimental conditions that are relevant for physiological function. The proposed approach is generally applicable to many IDPs/IDRs whose assignment is available in the Biological Magnetic Resonance Bank (BMRB) to investigate how their properties are modulated by different, physiologically relevant conditions. The experiments, tested on α-synuclein, are then used to investigate how α-synuclein senses Ca

Crosstalk between cellular pathways is often mediated through scaffold proteins that function as platforms for the assembly of signaling complexes. Based on yeast two-hybrid analysis, we report here the interaction between two complex scaffold proteins, CREB-binding protein (CBP) and the Ras GTPase-activating-like protein 1 (IQGAP1). Dissection of the interaction between the two proteins reveals that the central, thus far uncharacterized, region of IQGAP1 interacts with the HAT domain and the C-terminal intrinsically disordered region of CBP (termed ID5). Structural analysis of ID5 by solution NMR spectroscopy and SAXS reveals the presence of two regions with pronounced helical propensity. The ID5 region(s) involved in the interaction of nanomolar affinity were delineated by solution NMR titrations and pull-down assays. Moreover, we found that IQGAP1 acts as an inhibitor of the histone acetyltransferase (HAT) activity of CBP. In in vitro assays, the CBP-binding region of IQGAP1 positively and negatively regulates the function of HAT proteins of different families including CBP, KAT5 and PCAF. As many signaling pathways converge on CBP and IQGAP1, their interaction provides an interface between transcription regulation and the coordination of cytoskeleton. Disruption or alteration of the interaction between these scaffold proteins may lead to cancer development or metastatic processes, highlighting the importance of this interaction.

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). Electronic supplementary material The online version of this article (10.1007/s10858-020-00301-5) contains supplementary material, which is available to authorized users.

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.

The increasingly recognized biological relevance of intrinsically disordered proteins requires a continuous expansion of the tools for their characterization via NMR spectroscopy, the only technique so far able to provide atomic-resolution information on these highly mobile macromolecules. Here we present the implementation of projection spectroscopy in 13C-direct detected NMR experiments to achieve the sequence specific assignment of IDPs. The approach was used to obtain the complete backbone assignment at high temperature of α-synuclein, a paradigmatic intrinsically disordered protein.

A small molecule binds to a disordered protein in its monomeric form, preventing its aggregation linked to Alzheimer’s disease., Disordered proteins are challenging therapeutic targets, and no drug is currently in clinical use that modifies the properties of their monomeric states. Here, we identify a small molecule (10074-G5) capable of binding and sequestering the intrinsically disordered amyloid-β (Aβ) peptide in its monomeric, soluble state. Our analysis reveals that this compound interacts with Aβ and inhibits both the primary and secondary nucleation pathways in its aggregation process. We characterize this interaction using biophysical experiments and integrative structural ensemble determination methods. We observe that this molecule increases the conformational entropy of monomeric Aβ while decreasing its hydrophobic surface area. We also show that it rescues a Caenorhabditis elegans model of Aβ-associated toxicity, consistent with the mechanism of action identified from the in silico and in vitro studies. These results illustrate the strategy of stabilizing the monomeric states of disordered proteins with small molecules to alter their behavior for therapeutic purposes.

Henipaviruses are severe human pathogens responsible for severe encephalitis. Their V protein is a key player in the evasion of the host innate immune response. We have previously reported a biophysical characterization of the Henipavirus V proteins and shown that they interact with DDB1, a cellular protein that is a component of the ubiquitin ligase E3 complex. Here, we serendipitously discovered that the Hendra virus V protein undergoes a liquidhydrogel phase transition. By combining experimental and bioinformatics approaches, we have identified the V region responsible for this phenomenon. This region (referred to as PNT3), which falls within the long intrinsically disordered region of V, was further investigated using a combination of biophysical and structural approaches. ThioflavinT and Congo red binding assays, together with negative-staining electron microscopy studies, show that this region forms amyloid-like, β-enriched structures. Such structures are also formed in mammal cells transfected to express PNT3. Those cells also exhibit a reduced viability in the presence of a stress agent. Interestingly, mammal cells expressing a rationally designed, non-amyloidogenic PNT3 variant (PNT33A), appear to be much less sensitive to the stress agent, thus enabling the establishment of a link between fibril formation and cell toxicity. The present findings therefore pinpoint a so far never reported possible mechanism of virus-induced cell toxicity.

Molecular chaperones such as Hsp40 and Hsp70 hold the androgen receptor (AR) in an inactive conformation. They are released in the presence of androgens, enabling transactivation and causing the receptor to become aggregation-prone. Here we show that these molecular chaperones recognize a region of the AR N-terminal domain (NTD), including a FQNLF motif, that interacts with the AR ligand-binding domain (LBD) upon activation. This suggests that competition between molecular chaperones and the LBD for the FQNLF motif regulates AR activation. We also show that, while the free NTD oligomerizes, binding to Hsp70 increases its solubility. Stabilizing the NTD-Hsp70 interaction with small molecules reduces AR aggregation and promotes its degradation in cellular and mouse models of the neuromuscular disorder spinal bulbar muscular atrophy. These results help resolve the mechanisms by which molecular chaperones regulate the balance between AR aggregation, activation and quality control.

Disordered proteins are challenging therapeutic targets, and no drug is currently in clinical use that has been shown to modify the properties of their monomeric states. Here, we identify a small molecule capable of binding and sequestering the amyloid-β peptide (Aβ) in its monomeric, soluble state. Our analysis reveals that this compound interacts with Aβ and inhibits both the primary and secondary nucleation pathways in its aggregation process. We characterise this interaction using biophysical experiments and integrative structural ensemble determination methods. We thus observe that this small molecule has the remarkable effect of increasing the conformational entropy of monomeric Aβ while decreasing its hydrophobic surface area. We then show that this small molecule rescues a Caenorhabditis elegans model of Aβ-associated toxicity in a manner consistent with the mechanism of action identified from the in silico and in vitro studies. These results provide an illustration of the strategy of targeting the monomeric states of disordered proteins with small molecules to alter their behaviour for therapeutic purposes.

Polyglutamine (polyQ) tracts are regions of low sequence complexity frequently found in transcription factors. Tract length often correlates with transcriptional activity and expansion beyond specific thresholds in certain human proteins is the cause of polyQ disorders. To study the structural basis of the association between tract length, transcriptional activity and disease, we addressed how the conformation of the polyQ tract of the androgen receptor, associated with spinobulbar muscular atrophy (SBMA), depends on its length. Here we report that this sequence folds into a helical structure stabilized by unconventional hydrogen bonds between glutamine side chains and main chain carbonyl groups, and that its helicity directly correlates with tract length. These unusual hydrogen bonds are bifurcate with the conventional hydrogen bonds stabilizing α-helices. Our findings suggest a plausible rationale for the association between polyQ tract length and androgen receptor transcriptional activity and have implications for establishing the mechanistic basis of SBMA. © 2019, The Author(s)., We thank Sandro Bottaro, Ernest Giralt, Gerhard Hummer, Víctor Muñoz, and Huan-Xiang Zhou for helpful discussions and the ICTS NMR facility, managed by the scientific and technological centers of the University of Barcelona (CCiT UB), for their help in NMR. K.L.-L. and M.B.A.K. acknowledge funding from the Lundbeck Foundation and the BRAINSTRUC initiative. B.T. and J.A. acknowledge, respectively, FPI and Juan de la Cierva fellowships from MINECO. R.P and I.C.F. acknowledge funding from the European Commission (iNEXT, 653706). M.O. acknowledges the Spanish Ministry of Science (BFU2014-61670-EXP), the Catalan SGR, the Instituto Nacional de Bioinformática, the Biomolecular and Bioinformatics Resources Platform (ISCIII PT 13/0001/0030) co-funded by the Fondo Europeo de Desarrollo Regional (FEDER). R.C. acknowledges funding from MINECO (CTQ2016-78636-P). X.S. acknowledges funding from AGAUR (2017 SGR 324), Marató TV3 (102030), MINECO (BIO2012-31043 and BIO2015-70092-R), and the European Research Council (CONCERT, contract number 648201). IRB Barcelona is the recipient of a Severo Ochoa Award of Excellence from MINECO (Government of Spain)

Growing evidence implicates α-synuclein aggregation as a key driver of neurodegeneration in Parkinson’s disease (PD) and other neurodegenerative disorders. Herein, the molecular and structural mechanisms of inhibiting α-synuclein aggregation by novel analogs of nordihydroguaiaretic acid (NDGA), a phenolic dibenzenediol lignan, were explored using an array of biochemical and biophysical methodologies. NDGA analogs induced modest, progressive compaction of monomeric α-synuclein, preventing aggregation into amyloid-like fibrils. This conformational remodeling preserved the dynamic adoption of α-helical conformations, which are essential for physiological membrane interactions. Oxidation-dependent NDGA cyclization was required for the interaction with monomeric α-synuclein. NDGA analog-pretreated α-synuclein did not aggregate even without NDGA-analogs in the aggregation mixture. Strikingly, NDGA-pretreated α-synuclein suppressed aggregation of naïve untreated aggregation-competent monomeric α-synuclein. Further, cyclized NDGA reduced α-synuclein-driven neurodegeneration in Caenorhabditis elegans. The cyclized NDGA analogs may serve as a platform for the development of small molecules that stabilize aggregation-resistant α-synuclein monomers without interfering with functional conformations yielding potential therapies for PD and related disorders.

The small-DNA human adenovirus encodes one of the most versatile molecular hubs, the E1A protein. This protein is essential for productive viral infection in human cells and a vast amount of biologically relevant data are available on its interactions with host proteins. Up to now, however, no high-resolution structural and dynamic information on E1A is available despite its important biological role. Among the different spliced variants of E1A, two are expressed at high level in the early stage of infection. These are 243 and 289 residues isoforms. Herein, we present their NMR characterization, showing that they are both highly disordered, but also demonstrate a certain heterogeneous behavior in terms of structural and dynamic properties. Furthermore, we present the characterization of the isolated domain of the longer variant, known as CR3. This study opens the way to understanding at the molecular level how E1A functions.

Intrinsically disordered proteins (IDPs) as well as intrinsically disordered regions (IDRs) of complex protein machineries have recently been recognized as key players in many cellular functions. NMR represents a unique tool to access atomic resolution structural and dynamic information on highly flexible IDPs/IDRs. Improvements in instrumental sensitivity made heteronuclear direct detection possible for biomolecular NMR applications. The CON experiment has become one of the most useful NMR experiments to get a snapshot of an IDP/IDR in conditions approaching physiological ones. The availability of NMR spectrometers equipped with multiple receivers now enables the acquisition of several experiments simultaneously instead of one after the other. Here, we propose several variants of the CON experiment in which, during the recovery delay, a second two-dimensional experiment is acquired, either based on 1H detection (CON//HN) or on 15N detection (CON//btNH, CON//(H)CAN). The possibility to collect simultaneous snapshots of an IDP/IDR through different two-dimensional spectra provides a novel tool to follow chemical reactions, such as the occurrence of posttranslational modifications, as well as to study samples of limited lifetime such as cell lysates or whole cells.

Polyglutamine (polyQ) tracts are regions of low sequence complexity of variable length found in more than one hundred human proteins. These tracts are frequent in activation domains of transcription factors and their length often correlates with transcriptional activity. In addition, in nine proteins, tract elongation beyond specific thresholds causes polyQ disorders. To study the structural basis of the association between tract length, transcriptional activity and disease, here we addressed how the conformation of the polyQ tract of the androgen receptor (AR), a transcription factor associated with the polyQ disease spinobulbar muscular atrophy (SBMA), depends on its length. We found that the tract folds into a helical structure stabilized by unconventional hydrogen bonds between glutamine side chains and main chain carbonyl groups. These bonds are bifurcate with the conventional main chain to main chain hydrogen bonds stabilizing α-helices. In addition, since tract elongation provides additional interactions, the helicity of the polyQ tract directly correlates with its length. These findings suggest a plausible rationale for the association between polyQ tract length and AR transcriptional activity and have implications for establishing the mechanistic basis of SBMA.

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

NMR spectroscopy is one of the main techniques used for high-resolution studies of intrinsically disordered proteins (IDPs), permitting mapping of the structural and dynamic features of all the amino acids constituting the polypeptide at atomic resolution. Only proline residues are less straightforward to characterize because they lack any amide proton, thus rendering them not directly visible in the commonly used 2D 1 H,15 N correlation experiments. However, proline residues are highly abundant in IDPs and can mediate important functions. In this work we present an easy and effective way to obtain fingerprints of proline residues in IDPs at high resolution.

Here, we present a structural and dynamic description of CBP-ID4 at atomic resolution. ID4 is the fourth intrinsically disordered linker of CREB-binding protein (CBP). In spite of the largely disordered nature of CBP-ID4, NMR chemical shifts and relaxation measurements show a significant degree of α-helix sampling in the protein regions encompassing residues 2-25 and 101-128 (1852-1875 and 1951-1978 in full-length CBP). Proline residues are uniformly distributed along the polypeptide, except for the two α-helical regions, indicating that they play an active role in modulating the structural features of this CBP fragment. The two helical regions are lacking known functional motifs, suggesting that they represent thus-far uncharacterized functional modules of CBP. This work provides insights into the functions of this protein linker that may exploit its plasticity to modulate the relative orientations of neighboring folded domains of CBP and fine-tune its interactions with a multitude of partners.

Spin-state-selective methods to achieve homonuclear decoupling in the direct acquisition dimension of 13C detected NMR experiments have been one of the key contributors to converting 13C detected NMR experiments into really useful tools for studying biomolecules. We discuss here in detail the various methods that have been proposed, summarize the large array of new experiments that have been developed and present applications to different kinds of proteins in different aggregation states.

International audience; The Achaete-scute homolog 1 (Ascl1) protein regulates a large subset of genes that leads neuronal progenitor cells to distinctive differentiation pathways during human brain development. Although it is well known that Ascl1 binds DNA as a homo- or heterodimer via its basic helix-loop-helix (bHLH) motif, little is known about the conformational sampling properties of the DNA-free full-length protein, and in particular about the bHLH domain-flanking N- and C-terminal segments, which are predicted to be highly disordered in solution. The structural heterogeneity, low solubility, and high aggregation propensity of Ascl1 in aqueous buffer solutions make high-resolution studies of this protein a challenging task. Here, we have adopted a fragment-based strategy that allowed us to obtain high-quality NMR data providing, to our knowledge, the first comprehensive high-resolution information on the structural propensities and conformational dynamics of Ascl1. The emerging picture is that of an overall extended and highly dynamic polypeptide chain comprising three helical segments and lacking persistent long-range interactions. We also show that the C-terminal helix of the bHLH domain is involved in intermolecular interactions, even in the absence of DNA. Our results contribute to a better understanding of the mechanisms of action that govern the regulation of proneural transcription factors.

International audience; Using SAXS and NMR spectroscopy, we herein provide a high-resolution description of the intrinsically disordered N-terminal domain (PNT, aa 1-406) shared by the Nipah virus (NiV) phosphoprotein (P) and V protein, two key players in viral genome replication and in evasion of the host innate immune response, respectively. The use of multidimensional NMR spectroscopy allowed us to assign as much as 91% of the residues of this intrinsically disordered domain whose size constitutes a technical challenge for NMR studies. Chemical shifts and nuclear relaxation measurements provide the picture of a highly flexible protein. The combination of SAXS and NMR information enabled the description of the conformational ensemble of the protein in solution. The present results, beyond providing an overall description of the conformational behavior of this intrinsically disordered region, also constitute an asset for obtaining atomistic information in future interaction studies with viral and/or cellular partners. The present study can thus be regarded as the starting point towards the design of inhibitors that by targeting crucial protein-protein interactions involving PNT might be instrumental to combat this deadly virus. The Nipah virus (NiV), together with its close relative Hendra virus (HeV), is a zoonotic paramyxovirus responsible for severe encephalitis in humans. The NiV and HeV have been classified in the Henipavirus genus 1 that also comprises the later on discovered Cedar virus (CedV) 2. Because of their high pathogenic power, broad host range, high interspecies transmission and lack of therapeutics and vaccines, henipaviruses are classified as bio-security level 4 (BSL-4) pathogens and are considered as potential bio-terrorism agents. The genome of henipaviruses is made of a non-segmented, single-stranded RNA molecule of negative polarity that is encapsidated by a regular array of nucleoprotein (N) monomers to form a helical nucleocapsid. This N:RNA complex, and not naked RNA, is the substrate used by the viral polymerase for both transcription and replication. The viral polymerase is a complex consisting of the large (L) protein, which bears all the enzymatic activities, and the phosphoprotein (P). Through its interaction with both L and the nucleocapsid, the P protein acts as a tether and recruits L onto the N:RNA template. In addition, P also serves as a chaperon for both L 3 and N in that it is required for proper folding/maturation of L and maintains N in a monomeric, RNA-free form 4. Therefore P is a pivotal protein endowed with multiple functions critical for both transcription and replication. The repertoire of P functions is further expanded by the peculiar coding capacity of the P gene. Indeed, beyond the P protein, the P gene also codes for the V and W proteins that are generated through the addition of either one (protein V) or two (protein W) non-templated guanosines at the editing site of the P messenger. The addition of these guanosines triggers a downstream frame-shift. The P, V, and W proteins therefore share a OPEN