Structural Insights into the Dimeric Form of Bacillus subtilis RNase Y Using NMR and AlphaFold.

RNase Y is a crucial component of genetic translation, acting as the key enzyme initiating mRNA decay in many Gram-positive bacteria. The N-terminal domain of Bacillus subtilis RNase Y (Nter-BsRNaseY) is thought to interact with various protein partners within a degradosome complex. Bioinformatics and biophysical analysis have previously shown that Nter-BsRNaseY, which is in equilibrium between a monomeric and a dimeric form, displays an elongated fold with a high content of α-helices. Using multidimensional heteronuclear NMR and AlphaFold models, here, we show that the Nter-BsRNaseY dimer is constituted of a long N-terminal parallel coiled-coil structure, linked by a turn to a C-terminal region composed of helices that display either a straight or bent conformation. The structural organization of the N-terminal domain is maintained within the AlphaFold model of the full-length RNase Y, with the turn allowing flexibility between the N- and C-terminal domains. The catalytic domain is globular, with two helices linking the KH and HD modules, followed by the C-terminal region. This latter region, with no function assigned up to now, is most likely involved in the dimerization of B. subtilis RNase Y together with the N-terminal coiled-coil structure.

Optimisation of spatially-encoded diffusion-ordered NMR spectroscopy for the analysis of mixtures.

Diffusion-ordered NMR spectroscopy (DOSY NMR) is a widely used method for the analysis of mixtures. It can be used to separate the spectra of a mixture's components and to analyse interactions. The classic implementation of DOSY experiments, based on an incrementation of the diffusion-encoding gradient area, requires several minutes or more to collect a 2D data set. Spatially-encoded (SPEN) DOSY makes it possible to collect a complete data set in less than 1 s, by spatial parallelisation of the effective gradient area. While several short descriptions of SPEN DOSY experiments have been reported, a thorough characterisation of its features and its practical use is missing, and this hinders the use of the method. Here, we present the unusual principles and implementation of the SPEN DOSY experiment, an understanding of which is useful to make optimal use of the method. The encoding and acquisition steps are described, and the parameter relations that govern the setup of SPEN DOSY experiments are discussed. The influence of key parameters, including on sensitivity, is illustrated experimentally on mixtures of small molecules. This study should be useful for the setup of SPEN DOSY experiments, which are particularly useful for systems that evolve in time.

Expanding the Disorder-Function Paradigm in the C-Terminal Tails of Erbbs.

ErbBs are receptor tyrosine kinases involved not only in development, but also in a wide variety of diseases, particularly cancer. Their extracellular, transmembrane, juxtamembrane, and kinase folded domains were described extensively over the past 20 years, structurally and functionally. However, their whole C-terminal tails (CTs) following the kinase domain were only described at atomic resolution in the last 4 years. They were shown to be intrinsically disordered. The CTs are known to be tyrosine-phosphorylated when the activated homo- or hetero-dimers of ErbBs are formed. Their phosphorylation triggers interaction with phosphotyrosine binding (PTB) or Src Homology 2 (SH2) domains and activates several signaling pathways controling cellular motility, proliferation, adhesion, and apoptosis. Beyond this passive role of phosphorylated domain and site display for partners, recent structural and function studies unveiled active roles in regulation of phosphorylation and interaction: the CT regulates activity of the kinase domain; different phosphorylation states have different compaction levels, potentially modulating the succession of phosphorylation events; and prolines have an important role in structure, dynamics, and possibly regulatory interactions. Here, we review both the canonical role of the disordered CT domains of ErbBs as phosphotyrosine display domains and the recent findings that expand the known range of their regulation functions linked to specific structural and dynamic features.

ß-Hairpin Peptide Mimics Decrease Human Islet Amyloid Polypeptide (hIAPP) Aggregation.

Amyloid diseases are degenerative pathologies, highly prevalent today because they are closely related to aging, that have in common the erroneous folding of intrinsically disordered proteins (IDPs) which aggregate and lead to cell death. Type 2 Diabetes involves a peptide called human islet amyloid polypeptide (hIAPP), which undergoes a conformational change, triggering the aggregation process leading to amyloid aggregates and fibers rich in ß-sheets mainly found in the pancreas of all diabetic patients. Inhibiting the aggregation of amyloid proteins has emerged as a relevant therapeutic approach and we have recently developed the design of acyclic flexible hairpins based on peptidic recognition sequences of the amyloid ß peptide (Aß1-42) as a successful strategy to inhibit its aggregation involved in Alzheimer's disease. The present work reports the extension of our strategy to hIAPP aggregation inhibitors. The design, synthesis, conformational analyses, and biophysical evaluations of dynamic ß-hairpin like structures built on a piperidine-pyrrolidine ß-turn inducer are described. By linking to this ß-turn inducer three different arms (i) pentapeptide, (ii) tripeptide, and (iii) α/aza/aza/pseudotripeptide, we demonstrate that the careful selection of the peptide-based arms from the sequence of hIAPP allowed to selectively modulate its aggregation, while the peptide character can be decreased. Biophysical assays combining, Thioflavin-T fluorescence, transmission electronic microscopy, capillary electrophoresis, and mass spectrometry showed that the designed compounds inhibit both the oligomerization and the fibrillization of hIAPP. They are also capable to decrease the aggregation process in the presence of membrane models and to strongly delay the membrane-leakage induced by hIAPP. More generally, this work provides the proof of concept that our rational design is a versatile and relevant strategy for developing efficient and selective inhibitors of aggregation of amyloidogenic proteins.

Structural and dynamic characterization of the C-terminal tail of ErbB2: Disordered but not random.

ErbB2 (or HER2) is a receptor tyrosine kinase overexpressed in some breast cancers and associated with poor prognosis. Treatments targeting the receptor extracellular and kinase domains have greatly improved disease outcome in the last 20 years. In parallel, the structures of these domains have been described, enabling better mechanistic understanding of the receptor function and targeted inhibition. However, the ErbB2 disordered C-terminal cytoplasmic tail (CtErbB2) remains very poorly characterized in terms of structure, dynamics, and detailed functional mechanism. Yet, it is where signal transduction is triggered via phosphorylation of tyrosine residues and carried out via interaction with adaptor proteins. Here, we report the first description, to our knowledge, of the ErbB2 disordered tail at atomic resolution using NMR, complemented by small-angle x-ray scattering. We show that although no part of CtErbB2 has any fully populated secondary or tertiary structure, it contains several transient α-helices and numerous transient polyproline II helices, populated up to 20 and 40%, respectively, and low but significant compaction. The presence of some structural elements suggests, along the lines of the results obtained for EGFR (ErbB1), that they may have a functional role in ErbB2's autoregulation processes. In addition, the transient formation of polyproline II helices is compliant with previously suggested interactions with SH3 domains. All in all, our in-depth structural study opens perspectives in the mechanistic understanding of ErbB2.

Fluorinated Triazole Foldamers: Folded or Extended Conformational Preferences.

The Ministère de l'Enseignement Supérieur et de la Recherche (MESR) is thanked for financial support for José Laxio Arenas. The China Scholarship Council is thanked for financial support for Yaochun Xu. The authors thank Pr. Vadim Soloshonok and TOSOH F-TECH, Inc. for the kind gift of N-terbutyl-sulfinylimine.

NMR Characterization of the Influence of Zinc(II) Ions on the Structural and Dynamic Behavior of the New Delhi Metallo-ß-Lactamase-1 and on the Binding with Flavonols as Inhibitors.

New Delhi metallo-ß-lactamase-1 (NDM-1) has recently emerged as a global threat because of its ability to confer resistance to all common ß-lactam antibiotics. Understanding the molecular basis of ß-lactam hydrolysis by NDM is crucial for designing NDM inhibitors or ß-lactams resistant to their hydrolysis. In this study, for the first time, NMR was used to study the influence of Zn(II) ions on the dynamic behavior of NDM-1. Our results highlighted that the binding of Zn(II) in the NDM-1 active site induced several structural and dynamic changes on active site loop 2 (ASL2) and L9 loops and on helix α2. We subsequently studied the interaction of several flavonols: morin, quercetin, and myricetin were identified as natural and specific inhibitors of NDM-1. Quercetin conjugates were also synthesized in an attempt to increase the solubility and bioavailability. Our NMR investigations on NDM-1/flavonol interactions highlighted that both Zn(II) ions and the residues of the NDM-1 ASL1, ASL2, and ASL4 loops are involved in the binding of flavonols. This is the first NMR interaction study of NDM-1/inhibitors, and the models generated using HADDOCK will be useful for the rational design of more active inhibitors, directed against NDM-1.

Structural Characterization of N-WASP Domain V Using MD Simulations with NMR and SAXS Data.

Because of their large conformational heterogeneity, structural characterization of intrinsically disordered proteins (IDPs) is very challenging using classical experimental methods alone. In this study, we use NMR and small-angle x-ray scattering (SAXS) data with multiple molecular dynamics (MD) simulations to describe the conformational ensemble of the fully disordered verprolin homology domain of the neural Aldrich syndrome protein involved in the regulation of actin polymerization. First, we studied several back-calculation software of SAXS scattering intensity and optimized the adjustable parameters to accurately calculate the SAXS intensity from an atomic structure. We also identified the most appropriate force fields for MD simulations of this IDP. Then, we analyzed four conformational ensembles of neural Aldrich syndrome protein verprolin homology domain, two generated with the program flexible-meccano with or without NMR-derived information as input and two others generated by MD simulations with two different force fields. These four conformational ensembles were compared to available NMR and SAXS data for validation. We found that MD simulations with the AMBER-03w force field and the TIP4P/2005s water model are able to correctly describe the conformational ensemble of this 67-residue IDP at both local and global level.

Single-Scan Diffusion-Ordered NMR Spectroscopy of SABRE-Hyperpolarized Mixtures.

The analysis of complex mixtures of dissolved molecules is a major challenge, especially for systems that gradually evolve, e. g., in the course of a chemical reaction or in the case of chemical instability. 1D NMR is a fast and non-invasive method suitable for detailed molecular analysis, though of low sensitivity. Moreover, the spectral resolution of proton, the most commonly used and most sensitive stable isotope in NMR, is also quite limited. Spatially encoded (SPEN) experiments aim at creating in one acquisition a 2D data set by simultaneously performing different 1D sub-experiments on different slices of the NMR tube, at the price of an extra loss of sensitivity. Choosing translational diffusion coefficients as the additional dimension (the so-called DOSY approach) helps to recover proton spectra of each molecule in a mixture. The sensitivity limitation of SPEN NMR can, on the other hand, be addressed with hyperpolarization methods. Within hyperpolarization methods, signal amplification by reversible exchange (SABRE), based on parahydrogen, is the cheapest and the easiest one to set up, and allows multi-shot experiments. Here we show that the spectra of a mixture's components at millimolar concentration are resolved in few seconds by combining the SABRE, SPEN and DOSY concepts.

Modulation of the HIV nucleocapsid dynamics finely tunes its RNA-binding properties during virion genesis.

During HIV-1 assembly and budding, Gag protein, in particular the C-terminal domain containing the nucleocapsid domain (NCd), p1 and p6, is the site of numerous interactions with viral and cellular factors. Most in vitro studies of Gag have used constructs lacking p1 and p6. Here, using NMR spectroscopy, we show that the p1-p6 region of Gag (NCp15) is largely disordered, but interacts transiently with the NCd. These interactions modify the dynamic properties of the NCd. Indeed, using isothermal titration calorimetry (ITC), we have measured a higher entropic penalty to RNA-binding for the NCd precursor, NCp15, than for the mature form, NCp7, which lacks p1 and p6. We propose that during assembly and budding of virions, concomitant with Gag oligomerization, transient interactions between NCd and p1-p6 become salient and responsible for (i) a higher level of structuration of p6, which favours recruitment of budding partners; and (ii) a higher entropic penalty to RNA-binding at specific sites that favours non-specific binding of NCd at multiple sites on the genomic RNA (gRNA). The contributions of p6 and p1 are sequentially removed via proteolysis during Gag maturation such that the RNA-binding specificity of the mature protein is governed by the properties of NCd.

Spatially encoded diffusion-ordered NMR spectroscopy of reaction mixtures in organic solvents.

Diffusion-ordered NMR spectroscopy (DOSY) is a powerful method for the analysis of mixtures. Classic DOSY methods require several minutes of acquisition, and we show here that DOSY experiments can be recorded in less than one second for the challenging case of solution mixtures in low-viscosity solvents. The proposed method relies on a spatial encoding of the diffusion dimension, for which convection-compensation and spectral-selection strategies are introduced. The method is illustrated with the analysis of a reaction mixture, and more accurate estimates of the diffusion coefficients are obtained.

Structure-activity relationships of ß-hairpin mimics as modulators of amyloid ß-peptide aggregation.

Aggregation of amyloid proteins is currently involved in more than 20 serious human diseases that are actually untreated, such as Alzheimer's disease (AD). Despite many efforts made to target the amyloid cascade in AD, finding an aggregation inhibiting compound and especially modulating early oligomerization remains a relevant and challenging strategy. We report herein the first examples of small and non-peptide mimics of acyclic beta-hairpins, showing an ability to delay the fibrillization of amyloid-ß (Aß1-42) peptide and deeply modify its early oligomerization process. Modifications providing better druggability properties such as increased hydrophilicity and reduced peptidic character were performed. We also demonstrate that an appropriate balance between flexibility and stability of the ß-hairpin must be reached to adapt to the different shape of the various aggregated forms of the amyloid peptide. This strategy can be investigated to target other challenging amyloid proteins.

1H, 13C and 15N assignments of the C-terminal intrinsically disordered cytosolic fragment of the receptor tyrosine kinase ErbB2.

ErbB2 (or HER2) is a receptor tyrosine kinase that is involved in signaling pathways controlling cell division, motility and apoptosis. Though important in development and cell growth homeostasis, this protein, when overexpressed, participates in triggering aggressive HER2+ breast cancers. It is composed of an extracellular part and a transmembrane domain, both important for activation by dimerization, and a cytosolic tyrosine kinase, which activates its intrinsically disordered C-terminal end (CtErbB2). Little is known about this C-terminal part of 268 residues, despite its crucial role in interacting with adaptor proteins involved in signaling. Understanding its structural and dynamic characteristics could eventually lead to the design of new interaction inhibitors, and treatments complementary to those already targeting other parts of ErbB2. Here we report backbone and side-chain assignment of CtErbB2, which, together with structural predictions, confirms its intrinsically disordered nature.

Single-Scan 13 C Diffusion-Ordered NMR Spectroscopy of DNP-Hyperpolarised Substrates.

Diffusion-ordered NMR spectroscopy (DOSY) is a powerful approach for the analysis of molecular mixtures, yet its application range is limited by the relatively low sensitivity of NMR. We show here that spectrally resolved 13 C DOSY data can be collected, in a single scan, for substrates hyperpolarised by dissolution dynamic nuclear polarisation (D-DNP), which provides signal enhancements of several orders of magnitude. For this we use a convection-compensation pulse scheme, which we also analyse by numerical simulation. The proposed method further allows the acquisition of several consecutive DOSY spectra in a single D-DNP experiment.

Efficient simulation of ultrafast magnetic resonance experiments.

Magnetic resonance spectroscopy and imaging experiments in which spatial dynamics (diffusion and flow) closely coexists with chemical and quantum dynamics (spin-spin couplings, exchange, cross-relaxation, etc.) have historically been very hard to simulate - Bloch-Torrey equations do not support complicated spin Hamiltonians, and the Liouville-von Neumann formalism does not support explicit spatial dynamics. In this paper, we formulate and implement a more advanced simulation framework based on the Fokker-Planck equation. The proposed methods can simulate, without significant approximations, any spatio-temporal magnetic resonance experiment, even in situations when spatial motion co-exists intimately with quantum spin dynamics, relaxation and chemical kinetics.

Spatially encoded 2D and 3D diffusion-ordered NMR spectroscopy.

We show that the acquisition of 3D diffusion-ordered NMR spectroscopy (DOSY) experiments can be accelerated significantly with the use of spatial encoding (SPEN). The SPEN DOSY approach is discussed, analysed with numerical simulation, and illustrated on a mixture of small molecules.

Mutations in actin used for structural studies partially disrupt ß-thymosin/WH2 domains interaction.

Understanding the structural basis of actin cytoskeleton remodeling requires stabilization of actin monomers, oligomers, and filaments in complex with partner proteins, using various biochemical strategies. Here, we report a dramatic destabilization of the dynamic interaction with a model ß-thymosin/WH2 domain induced by mutations in actin. This result underlines that mutant actins should be used with prudence to characterize interactions with intrinsically disordered partners as destabilization of dynamic interactions, although identifiable by NMR, may be invisible to other structural techniques. It also highlights how both ß-thymosin/WH2 domains and actin tune local structure and dynamics in regulatory processes involving intrinsically disordered domains.

Robust and low cost uniform (15)N-labeling of proteins expressed in Drosophila S2 cells and Spodoptera frugiperda Sf9 cells for NMR applications.

Nuclear magnetic resonance spectroscopy is a powerful tool to study structural and functional properties of proteins, provided that they can be enriched in stable isotopes such as (15)N, (13)C and (2)H. This is usually easy and inexpensive when the proteins are expressed in Escherichiacoli, but many eukaryotic (human in particular) proteins cannot be produced this way. An alternative is to express them in insect cells. Labeled insect cell growth media are commercially available but at prohibitive prices, limiting the NMR studies to only a subset of biologically important proteins. Non-commercial solutions from academic institutions have been proposed, but none of them is really satisfying. We have developed a (15)N-labeling procedure based on the use of a commercial medium depleted of all amino acids and supplemented with a (15)N-labeled yeast autolysate for a total cost about five times lower than that of the currently available solutions. We have applied our procedure to the production of a non-polymerizable mutant of actin in Sf9 cells and of fragments of eukaryotic and viral membrane fusion proteins in S2 cells, which typically cannot be produced in E. coli, with production yields comparable to those obtained with standard commercial media. Our results support, in particular, the putative limits of a self-folding domain within a viral glycoprotein of unknown structure.

Structural features and interfacial properties of WH2, ß-thymosin domains and other intrinsically disordered domains in the regulation of actin cytoskeleton dynamics.

Many actin-binding proteins (ABPs) use complex multidomain architectures to integrate and coordinate multiple signals and interactions with the dynamic remodeling of actin cytoskeleton. In these proteins, small segments that are intrinsically disordered in their unbound native state can be functionally as important as identifiable folded units. These functional intrinsically disordered regions (IDRs) are however difficult to identify and characterize in vitro. Here, we try to summarize the state of the art in understanding the structural features and interfacial properties of IDRs involved in actin self-assembly dynamics. Recent structural and functional insights into the regulation of widespread, multifunctional WH2/ß-thymosin domains, and of other IDRs such as those associated with WASP/WAVE, formin or capping proteins are examined. Understanding the functional versatility of IDRs in actin assembly requires apprehending by multiple structural and functional approaches their large conformational plasticity and dynamics in their interactions. In many modular ABPs, IDRs relay labile interactions with multiple partners and act as interaction hubs in interdomain and protein-protein interfaces. They thus control multiple conformational transitions between the inactive and active states or between various active states of multidomain ABPs, and play an important role to coordinate the high turnover of interactions in actin self-assembly dynamics.

How a single residue in individual ß-thymosin/WH2 domains controls their functions in actin assembly.

ß-Thymosin (ßT) and WH2 domains are widespread, intrinsically disordered actin-binding peptides that display significant sequence variability and different regulations of actin self-assembly in motile and morphogenetic processes. Here, we reveal the structural mechanisms by which, in their 1:1 stoichiometric complexes with actin, they either inhibit assembly by sequestering actin monomers like Thymosin-ß4, or enhance motility by directing polarized filament assembly like Ciboulot ßT. We combined mutational, functional or structural analysis by X-ray crystallography, SAXS (small angle X-ray scattering) and NMR on Thymosin-ß4, Ciboulot, TetraThymosinß and the long WH2 domain of WASP-interacting protein. The latter sequesters G-actin with the same molecular mechanisms as Thymosin-ß4. Functionally different ßT/WH2 domains differ by distinct dynamics of their C-terminal half interactions with G-actin pointed face. These C-terminal interaction dynamics are controlled by the strength of electrostatic interactions with G-actin. At physiological ionic strength, a single salt bridge with actin located next to their central LKKT/V motif induces G-actin sequestration in both isolated long ßT and WH2 domains. The results open perspectives for elucidating the functions of ßT/WH2 domains in other modular proteins.