Search

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.

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.

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.

The SARS-CoV-2 nucleocapsid (N) protein is crucial for the highly organized packaging and transcription of the genomic RNA. Studying atomic details of the role of its intrinsically disordered regions (IDRs) in RNA recognition is challenging due to the absence of structure and to the repetitive nature of their primary sequence. IDRs are known to act in concert with the folded domains of N and here we use NMR spectroscopy to identify the priming events of N interacting with a regulatory SARS-CoV-2 RNA element. 13C-detected NMR experiments, acquired simultaneously to 1H detected ones, provide information on the two IDRs flanking the N-terminal RNA binding domain (NTD) within the N-terminal region of the protein (NTR, 1–248). We identify specific tracts of the IDRs that most rapidly sense and engage with RNA, and thus provide an atom-resolved picture of the interplay between the folded and disordered regions of N during RNA interaction.

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.

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.

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.

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.

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.

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.

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.

Intrinsically disordered proteins (IDPs) are characterized by highly flexible solvent exposed backbones and can sample many different conformations. These properties confer them functional advantages, complementary to those of folded proteins, which need to be characterized to expand our view of how protein structural and dynamic features affect function beyond the static picture of a single well defined 3D structure that has influenced so much our way of thinking. NMR spectroscopy provides a unique tool for the atomic resolution characterization of highly flexible macromolecules in general and of IDPs in particular. The peculiar properties of IDPs however have profound effects on spectroscopic parameters. It is thus worth thinking about these aspects to make the best use of the great potential of NMR spectroscopy to contribute to this fascinating field of research. In particular, after many years of dealing with exclusively heteronuclear NMR experiments based on (13)C direct detection, we would like here to address their relevance when studying IDPs.

The intracellular environment contains high concentrations of macromolecules occupying up to 30% of the total cellular volume. Presence of these macromolecules decreases the effective volume available for the proteins in the cell and thus increases the effective protein concentrations and stabilizes the compact protein conformations. Macromolecular crowding created by various macromolecules such as proteins, nucleic acids, and carbohydrates has been shown to have a significant effect on a variety of cellular processes including protein aggregation. Most studies of macromolecular crowding have used neutral, flexible polysaccharides that function primarily via excluded volume effect as model crowding agents. Here we have examined the effects of more rigid polysaccharides on protein structure and aggregation. Our results indicate that rigid and flexible polysaccharides influence protein aggregation via different mechanisms and suggest that, in addition to excluded volume effect, changes in solution viscosity and non-specific protein–polymer interactions influence the structure and dynamics of proteins in crowded environments.

The E7 protein from human papillomavirus (HPV) plays a key role in oncogenesis; for this reason, it is a target of great biomedical interest. To date, no high resolution information is available for the full protein. We present here the NMR characterization of the entire E7 from HPV16, one of the most oncogenic variants of the virus. The protein is very heterogeneous in terms of structural and dynamic properties with a highly flexible N-terminal module and a more structured C terminus. This opens possibilities for studies of molecular-level interactions and post-translational modifications of the protein to unravel functional details that might be linked to its highly oncogenic potential.

The multi-domain transcriptional coactivators CBP/p300 integrate a multitude of signaling inputs, interacting with more than 400 proteins via one or more of their globular domains. While CBP/p300 function is typically considered in terms of these structured domains, about half of the protein consists of intrinsically disordered regions (IDRs) of varying length. However, these IDRs have only been thought of as linkers that allow flexible spatial arrangement of the structured domains, but recent studies have shown that similar IDRs mediate specific and critical interactions in other proteins. To examine the roles of IDRs in CBP, we performed yeast-two-hybrid screenings of placenta and lung cancer cDNA libraries, which demonstrated that the long IDR linking the KIX domain and bromodomain of CBP (termed ID3) can potentially bind to several proteins. The RNA-binding Zinc-finger protein 106 (ZFP106) detected in both libraries was identified as a novel substrate for CBP-mediated acetylation. Nuclear magnetic resonance (NMR) spectroscopy combined with cross-linking experiments and competition-binding assays showed that the fully disordered isolated ID3 transiently interacts with an IDR of ZFP106 in a fashion that disorder of both regions is maintained. These findings demonstrate that beside the linking function, ID3 can also interact with acetylation substrates of CBP.

HPV-16 E7 is one of the key proteins that, by interfering with the host metabolism through many protein-protein interactions, hijacks cell regulation and contributes to malignancy. Here we report the high resolution investigation of the CR3 region of HPV-16 E7, both as an isolated domain and in the full-length protein. This opens the way to the atomic level study of the many interactions in which HPV-16 E7 is involved. Along these lines we show here the effect of one of the key post-translational modifications of HPV-16 E7, the phosphorylation by casein kinase II.

Thanks to recent fast progress, NMR is now in a strategic position to provide unique atomic resolution information on a variety of different biological macromolecules in different conditions (solution, solid state, in-cell). We would like here to present recent developments that enable to focus on intrinsically disordered proteins (IDPs) or intrinsically disordered regions (IDRs) of proteins of increasing size and complexity. They have attracted the attention of the scientific community challenging well accepted ideas and concepts and demanding an expansion of the structure function paradigm.

Expansions of polyglutamine (polyQ) tracts in nine different proteins cause a family of neurodegenerative disorders called polyQ diseases. Because polyQ tracts are potential therapeutic targets for these pathologies there is great interest in characterizing the conformations that they adopt and in understanding how their aggregation behavior is influenced by the sequences flanking them. We used solution NMR to study at single-residue resolution a 156-residue proteolytic fragment of the androgen receptor that contains a polyQ tract associated with the disease spinobulbar muscular atrophy, also known as Kennedy disease. Our findings indicate that a Leu-rich region preceding the polyQ tract causes it to become α-helical and appears to protect the protein against aggregation, which represents a new, to our knowledge, mechanism by which sequence context can minimize the deleterious properties of these repetitive regions. Our results have implications for drug discovery for polyQ diseases because they suggest that the residues flanking these repetitive sequences may represent viable therapeutic targets.

International audience; Intrinsically disordered proteins (IDPs) are functional proteins containing large fragments characterized by high local mobility. Bioinformatic studies have suggested that a significant fraction (more than 30%) of eukaryotic proteins has disordered regions of more than 50 amino acids in length. Hence, NMR methods for the characterization of local compactness and solvent accessibility in such highly disordered proteins are of high importance. Among the available approaches, the HET-SOFAST/BEST experiments (Schanda et al., 2006, Rennella et al., 2014) provide semi-quantitative information by monitoring longitudinal (1)H relaxation of amide protons under different initial conditions. However, when approaching physiological sample conditions, the potential of these amide (1)H detected experiments is reduced due to rapid amide proton solvent exchange. (13)C direct detection methods therefore provide a valuable alternative thanks to a higher chemical shift dispersion and their intrinsic insensitivity toward solvent exchange. Here we present two sets of (13)C-detected experiments, which indirectly measure (1)H(N) and (1)H(α) inversion recovery profiles. The experiments consist of an initial spin inversion-recovery block optimized for selective manipulation of different types of proton spins followed by a CON read-out scheme. The proposed experiments were tested on human α-synuclein and ubiquitin, two representative examples of unfolded and folded proteins.

Enzyme-inhibitor recognition is considered one of the most fundamental aspects in the area of drug discovery. However, the molecular mechanism of this recognition process (induced fit or prebinding and adaptive selection among multiple conformers) in several cases remains unexplored. In order to shed light toward this step of the recognition process in the case of human angiotensin I converting enzyme (hACE) and its inhibitor captopril, we have established a novel combinatorial approach exploiting solution NMR, flexible docking calculations, mutagenesis, and enzymatic studies. We provide evidence that an equimolar ratio of the cis and trans states of captopril exists in solution and that the enzyme selects only the trans state of the inhibitor that presents architectural and stereoelectronic complementarity with its substrate binding groove. (c) 2006 Elsevier Ltd. All rights reserved. Bioorganic and Medicinal Chemistry Letters

Carbon-13 direct detection NMR methods are feasible thanks to the improvements in probehead technology and to the development of new NMR experiments. We present here a complete set of experiments, based on C′ direct detection, developed to perform protein complete assignment of backbone and side-chains (except for aromatic rings). This strategy offers alternative solutions for demanding situations (paramagnetic and/or large molecules) and can be useful in general in conjunction with conventional experiments.

(13)C-(13)C NOESY experiments were performed under long mixing time conditions on reduced human superoxide dismutase (32 kDa, (15)N, (13C) and 70% (2)H labeled). (13)C-(13)C couplings were successfully eliminated through post-processing of in-phase-anti-phase (IPAP) data. It appears that at mixing time tau(m) of 3.0 s the spin diffusion mechanism allows the detection of 96% of the two-bond correlations involving C' and C(beta). The interpretation was confirmed by simulations. This approach broadens the range of applicability of (13)C-(13)C NOESY spectroscopy.

A large number of proteins carry out their function in highly flexible and disordered states, lacking a well-defined 3D structure. These proteins, referred to as intrinsically disordered proteins (IDPs), are now in the spotlight of modern structural biology. Nuclear magnetic resonance (NMR) spectroscopy represents a unique tool for accessing atomic resolution information on IDPs in complex environments as whole cells, provided that the methods are optimized to their peculiar properties and to the characteristics of in-cell experiments. We describe procedures for the preparation of in-cell NMR samples, as well as for the setup of NMR experiments and their application to in-cell studies, using human α-synuclein overexpressed in Escherichia coli as an example. The expressed protein is labeled with (13)C and (15)N stable isotopes to enable the direct recording of (13)C-detected NMR experiments optimized for the properties of IDPs. The entire procedure covers 24 h, including cell transformation, cell growth overnight, setup of the spectrometer and NMR experiment recording.

Intrinsically disordered proteins (IDPs) have recently attracted the attention of the scientific community challenging the well accepted structure–function paradigm. In the characterization of the dynamic features of proteins nuclear magnetic resonance spectroscopy (NMR) is a strategic tool of investigation. However the peculiar properties of IDPs, with the lack of a unique 3D structure and their high flexibility, have a strong impact on NMR observables (low chemical shift dispersion, efficient solvent exchange broadening) and thus on the quality of NMR spectra. Key aspects to be considered in the design of new NMR experiments optimized for the study of IDPs are discussed. A new experiment, based on direct detection of 13Cα, is proposed., This work has been supported in part by the Joint Research Activity and Access to Research Infrastructures (Bio–NMR, contract 261863) and by the Marie Curie ITN programs (IDPbyNMR, contract 264257) in the EC 7th Framework. S·S. acknowledges financial support for a short stay fellowship associated to Project CTQ2008–0080 from the Spanish Ministerio de Economía y Competitividad.

International audience; A double-zero quantum (DZQ)-refocused INADEQUATE experiment is introduced for J-based NMR correlations under ultra-fast (60 kHz) magic angle spinning (MAS). The experiment records two spectra in the same dataset, a double quantumsingle quantum (DQ-SQ) and zero quantumsingle quantum (ZQ-SQ) spectrum, whereby the corresponding signals appear at different chemical shifts in ?1. Furthermore, the spin-state selective excitation (S3E) J-decoupling block is incorporated in place of the second refocusing echo of the INADEQUATE scheme, providing an additional gain in sensitivity and resolution. The two sub-spectra acquired in this way can be treated separately by a shearing transformation, producing two diagonal-free single quantum (SQ-SQ)-type spectra, which are subsequently recombined to give an additional sensitivity enhancement, thus offering an improvement greater than a factor of two as compared to the original refocused INADEQUATE experiment. The combined DZQ scheme retains transverse magnetization on the initially polarized (I) spin, which typically exhibits a longer transverse dephasing time (T2') than its through-bond neighbour (S). By doing so, less magnetization is lost during the refocusing periods in the sequence to give even further gains in sensitivity for the J correlations. The experiment is demonstrated for the correlation between the carbonyl (CO) and alpha (CA) carbons in a microcrystalline sample of fully protonated, [15N,13C]-labelled CuII,?ZnII superoxide dismutase, and its efficiency is discussed with respect to other J-based schemes.

The characterization of intrinsically disordered proteins (IDPs) by NMR spectroscopy is made difficult by the extensive spectral overlaps. To overcome the intrinsic low-resolution of the spectra the introduction of high-dimensionality experiments is essential. We present here a set of high-resolution experiments based on direct (13)C-detection which proved useful in the assignment of α-synuclein, a paradigmatic IDP. In particular, we describe the implementation of 4D HCBCACON, HCCCON, HCBCANCO, 4/5D HNCACON and HNCANCO and 3/4D HCANCACO experiments, specifically tailored for spin system identification and backbone resonances sequential assignment. The use of non-uniform-sampling in the indirect dimension and of the H-flip approach to achieve longitudinal relaxation enhancement rendered the experiments very practical.

The title protein with MW 30,000 containing high-spin cobalt(II) has been thoroughly investigated through 1H NMR spectroscopy with the aid of selectively deuterated amino acids and 15N enrichment. The aim is that of showing the potentiality of the approach when local information by NMR is needed and the X-ray structure is available. The potential use of the pseudocontact shift is discussed; 90, 200, and 600 MHz spectrometers are used to investigate a spherical region at various distances from the metal ion. More than 35 signals of protons around cobalt(II) have been assigned.

The human small heat-shock protein αB-crystallin (αB) rescues misfolded proteins from irreversible aggregation during cellular stress. Binding of Cu(II) was shown to modulate the oligomeric architecture and the chaperone activity of αB. However, the mechanistic basis of this stimulation is so far not understood. We provide here first structural insights into this Cu(II)-mediated modulation of chaperone function using NMR spectroscopy and other biophysical approaches. We show that the α-crystallin domain is the elementary Cu(II)-binding unit specifically coordinating one Cu(II) ion with picomolar binding affinity. Putative Cu(II) ligands are His(83), His(104), His(111), and Asp(109) at the dimer interface. These loop residues are conserved among different metazoans, but also for human αA-crystallin, HSP20, and HSP27. The involvement of Asp(109) has direct implications for dimer stability, because this residue forms a salt bridge with the disease-related Arg(120) of the neighboring monomer. Furthermore, we observe structural reorganization of strands β2-β3 triggered by Cu(II) binding. This N-terminal region is known to mediate both the intermolecular arrangement in αB oligomers and the binding of client proteins. In the presence of Cu(II), the size and the heterogeneity of αB multimers are increased. At the same time, Cu(II) increases the chaperone activity of αB toward the lens-specific protein β(L)-crystallin. We therefore suggest that Cu(II) binding unblocks potential client binding sites and alters quaternary dynamics of both the dimeric building block as well as the higher order assemblies of αB.

Re-protonation is key: A combination of a high magnetic field (1 GHz) and ultra-fast magic-angle spinning (60 kHz) allows easy detection of NMR spectra revealing details of secondary and tertiary structures of medium-sized proteins. The technique was applied to the 153-residue microcrystalline Zn II-loaded superoxide dismutase (ZnII-SOD) fully [ 2H,13C,15N]-labeled and 100% re-protonated at the exchangeable sites. Copyright © 2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Here, we present a series of exclusively heteronuclear multidimensional NMR experiments, based on 13 C direct detection, which exploit the 1 H polarization as a starting source to increase the signal-to-noise ratio. This contributes to make this spectroscopy more useful and usable. Examples are reported for a suitable system such as securin, an intrinsically disordered protein of 22 kDa.

Keywords: NMR spectroscopy ; paramagnetic relaxation ; protein structures ; proton-driven spin diffusion ; structure elucidation Reference EPFL-ARTICLE-204398doi:10.1002/anie.200603093View record in Web of Science Record created on 2015-01-08, modified on 2017-05-12

Metal binding to the amyloid beta-peptide is suggested to be involved in the pathogenesis of Alzheimer's disease. We used high-resolution NMR to study zinc binding to amyloid beta-peptide 1-40 at physiologic pH. Metal binding induces a structural change in the peptide, which is in chemical exchange on an intermediate rate, between the apo-form and the holo-form, with respect to the NMR timescale. This causes loss of NMR signals in the resonances affected by the binding. Heteronuclear correlation experiments, (15)N-relaxation and amide proton exchange experiments on amyloid beta-peptide 1-40 revealed that zinc binding involves the three histidines (residues 6, 13 and 14) and the N-terminus, similar to a previously proposed copper-binding site [Syme CD, Nadal RC, Rigby SE, Viles JH (2004) J Biol Chem 279, 18169-18177]. Fluorescence experiments show that zinc shares a common binding site with copper and that the metals have similar affinities for amyloid beta-peptide. The dissociation constant K(d) of zinc for the fragment amyloid beta-peptide 1-28 was measured by fluorescence, using competitive binding studies, and that for amyloid beta-peptide 1-40 was measured by NMR. Both methods gave K(d) values in the micromolar range at pH 7.2 and 286 K. Zinc also has a second, weaker binding site involving residues between 23 and 28. At high metal ion concentrations, the metal-induced aggregation should mainly have an electrostatic origin from decreased repulsion between peptides. At low metal ion concentrations, on the other hand, the metal-induced structure of the peptide counteracts aggregation.

beta-parvalbumin, also known as oncomodulin, is a small Ca2+-binding protein belonging to the so-called EF-hand family. At variance with the ubiquitary a-parvalbumin, the b-form is almost exclusively expressed in tumor cells. The solution structure of the recombinant human protein has been solved by us (Babini et al. 2004) mostly based on proton assignment. As this protein is a potential drug target, its full assignment may be of general interest per se.We present here its virtually complete assignment, obtained with the aid of 13C- direct detection 2D experiments, including a novel pulse sequence designed for the assignment of aromatic Cc nuclei (see Supplementary material). All backbone resonances are assigned except for the HN and N of M1. Still unassigned are the N terminal in Lys residues, the ring NH and N signals of H108, and the guanidine signals of R20 and R49. In summary, 93.7% 1H, 99.4% 13C and 89.8% 15N of the total protein nuclei were assigned.

Copper(II) and platinum(II) complexes of 2-benzoylpyrrole (2-BZPH) were synthesized and characterized with IR, 1 H and 1 3 C NMR spectroscopies and coordination geometry with ligands arranged in transoid fashion. The crystal structure of [Cu I I (2-BZP) 2 ] was determined by X-ray diffraction. Death of complex treated Jurkat cells was measured by flow cytometry. The bis-chelate complexes [Cu I I (2-BZP) 2 ] and [Pt I I (2-BZP) 2 ] adopt square-planar coordination geometry with ligands, arranged in transoid fashion. Concentrations of 1-10 μM Platinum(II) complexes reduced cell survival from 100% to 20%, in contrast to the copper(II) complex which caused no cell death at a concentration of 10 μM. While the Pt I I complexes may have damaged DNA to induce cell death. treatment with the Cull complex did not induce Jurkat cell death.

We report the design and synthesis through solid phase 9-flourenylmethoxycarbonyl (Fmoc) chemistry of the two angiotensin-I converting enzyme active sites possessing the general sequence HEMGHX23EAIGDX3. Their zinc-binding properties were monitored in solution through high-resolution 1H-NMR. The obtained data were analyzed in terms of chemical shift differences. The results indicate that zinc binds to the HEMGH and the EAIGD characteristic motifs, and suggest possible coordination modes of zinc in the native enzyme. © 2003 Wiley Periodicals, Inc. Biopolymers 69: 244–252, 2003

13 C, 17 O and 57 Fe NMR spectra of several carbonmonoxy hemoprotein models with varying polar and steric effects of the distal organic superstructure, constraints of the proximal side, and porphyrin ruffling are reported. Both heme models and heme proteins obey a similar excellent linear δ ( 13 C) versus ν (C–O) relationship which is primarily due to modulation of π-back-bonding from the Fe d π to CO π * orbital by the distal pocket polar interactions. The lack of correlation between δ ( 13 C) and δ ( 17 O) suggests that the two probes do not reflect a similar type of electronic and structural perturbation. δ ( 17 O) is not primarily influenced by the local distal field interactions and does not correlate with any single structural property of the Fe–C–O unit; however, atropisomerism and deformation of the porphyrin geometry appear to play a significant role. 57 Fe shieldings vary by nearly 900 ppm among various hemes and an excellent correlation was found between δ ( 57 Fe) and the absolute crystallographic average displacement of the meso carbon atoms, | C m |, relative to the porphyrin core mean plane. The excellent correlation between iron-57 shieldings and the average shieldings of the meso carbons of the porphyrin skeleton of TPP derivatives suggests that the two probes reflect a similar type of electronic and structural perturbation which is primarily porphyrin ruffling.

Paramagnetic NMR data can be profitably incorporated in structural refinement protocols of metalloproteins or metal-substituted proteins, mostly as distance or angle restraints. However, they could in principle provide much more information, because the magnetic susceptibility of a paramagnetic metal ion is largely determined by its coordination sphere. This information can in turn be used to evaluate changes occurring in the coordination sphere of the metal when ligands (e.g.: inhibitors) are bound to the protein. This gives an experimental handle on the molecular structure in the vicinity of the metal which falls in the so-called blind sphere. The magnetic susceptibility anisotropy tensors of cobalt(II) and nickel(II) ions bound to human carbonic anhydrase II in free and inhibited forms have been determined. The change of the magnetic susceptibility anisotropy is directly linked to the binding mode of different ligands in the active site of the enzyme. Indication about the metal coordination sphere in the presence of an inhibitor in pharmaceutically relevant proteins could be important in the design of selective drugs with a structure-based approach.

12 pags., 4 figs., 3 tabs., SARS-CoV-2 (SCoV2) and its variants of concern pose serious challenges to the public health. The variants increased challenges to vaccines, thus necessitating for development of new intervention strategies including anti-virals. Within the international Covid19-NMR consortium, we have identified binders targeting the RNA genome of SCoV2. We established protocols for the production and NMR characterization of more than 80 % of all SCoV2 proteins. Here, we performed an NMR screening using a fragment library for binding to 25 SCoV2 proteins and identified hits also against previously unexplored SCoV2 proteins. Computational mapping was used to predict binding sites and identify functional moieties (chemotypes) of the ligands occupying these pockets. Striking consensus was observed between NMR-detected binding sites of the main protease and the computational procedure. Our investigation provides novel structural and chemical space for structure-based drug design against the SCoV2 proteome., Work at BMRZ is supported by the state of Hesse. Work in Covid19-NMR was supported by the Goethe Corona Funds, by the IWBEFRE-program 20007375 of state of Hesse, the DFG through CRC902: “Molecular Principles of RNA-based regulation.” and through infrastructure funds (project numbers: 277478796, 277479031, 392682309, 452632086, 70653611) and by European Union’s Horizon 2020 research and innovation program iNEXT-discovery under grant agreement No 871037. BY-COVID receives funding from the European Union’s Horizon Europe Research and Innovation Programme under grant agreement number 101046203. “INSPIRED” (MIS 5002550) project, implemented under the Action “Reinforcement of the Research and Innovation Infrastructure,” funded by the Operational Program “Competitiveness, Entrepreneurship and Innovation” (NSRF 2014–2020) and co-financed by Greece and the EU (European Regional Development Fund) and the FP7 REGPOT CT-2011-285950—“SEE-DRUG” project (purchase of UPAT’s 700 MHz NMR equipment). The support of the CERM/CIRMMP center of Instruct-ERIC is gratefully acknowledged. This work has been funded in part by a grant of the Italian Ministry of University and Research (FISR2020IP_02112, ID-COVID) and by Fondazione CR Firenze. A.S. is supported by the Deutsche Forschungsgemeinschaft [SFB902/B16, SCHL2062/2-1] and the Johanna Quandt Young Academy at Goethe [2019/AS01]. M.H. and C.F. thank SFB902 and the Stiftung Polytechnische Gesellschaft for the Scholarship. L.L. work was supported by the French National Research Agency (ANR, NMR-SCoV2-ORF8), the Fondation de la Recherche Médicale (FRM, NMR-SCoV2-ORF8), FINOVI and the IR-RMN-THC Fr3050 CNRS. Work at UConn Health was supported by grants from the US National Institutes of Health (R01 GM135592 to B.H., P41 GM111135 and R01 GM123249 to J.C.H.) and the US National Science Foundation (DBI 2030601 to J.C.H.). Latvian Council of Science Grant No. VPP-COVID-2020/1-0014. National Science Foundation EAGER MCB-2031269. This work was supported by the grant Krebsliga KFS-4903-08-2019 and SNF-311030_192646 to J.O. P.G. (ITMP) The EOSC Future project is co-funded by the European Union Horizon Programme call INFRAEOSC-03-2020—Grant Agreement Number 101017536. Open Access funding enabled and organized by Projekt DEAL

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., Hsp chaperones stabilize the inactive conformation of androgen receptor (AR) and are released upon hormone-induced AR activation. Here, the authors locate the Hsp binding region on AR, and show that Hsp70 reduces AR aggregation and promotes AR degradation in cellular and mouse models of a neuromuscular disorder.