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The structural investigation of intrinsically disordered proteins (IDPs) requires ensemble models describing the diversity of the conformational states of the molecule. Due to their probabilistic nature, there is a need for new paradigms that understand and treat IDPs from a purely statistical point of view, considering their conformational ensembles as well-defined probability distributions. In this work, we define a conformational ensemble as an ordered set of probability distributions and provide a suitable metric to detect differences between two given ensembles at the residue level, both locally and globally. The underlying geometry of the conformational space is properly integrated, one ensemble being characterized by a set of probability distributions supported on the three-dimensional Euclidean space (for global-scale comparisons) and on the two-dimensional flat torus (for local-scale comparisons). The inherent uncertainty of the data is also taken into account to provide finer estimations of the differences between ensembles. Additionally, an overall distance between ensembles is defined from the differences at the residue level. We illustrate the potential of the approach with several examples of applications for the comparison of conformational ensembles: (i) produced from molecular dynamics (MD) simulations using different force fields, and (ii) before and after refinement with experimental data. We also show the usefulness of the method to assess the convergence of MD simulations, and discuss other potential applications such as in machine-learning-based approaches. The numerical tool has been implemented in Python through easy-to-use Jupyter Notebooks available at https://gitlab.laas.fr/moma/WASCO., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier Ltd. All rights reserved.)

Von Willebrand Factor (vWF), a 300-kDa plasma protein key to homeostasis, is cleaved at a single site by multi-domain metallopeptidase ADAMTS-13. vWF is the only known substrate of this peptidase, which circulates in a latent form and becomes allosterically activated by substrate binding. Herein, we characterised the complex formed by a competent peptidase construct (AD13-MDTCS) comprising metallopeptidase (M), disintegrin-like (D), thrombospondin (T), cysteine-rich (C), and spacer (S) domains, with a 73-residue functionally relevant vWF-peptide, using nine complementary techniques. Pull-down assays, gel electrophoresis, and surface plasmon resonance revealed tight binding with sub-micromolar affinity. Cross-linking mass spectrometry with four reagents showed that, within the peptidase, domain D approaches M, C, and S. S is positioned close to M and C, and the peptide contacts all domains. Hydrogen/deuterium exchange mass spectrometry revealed strong and weak protection for C/D and M/S, respectively. Structural analysis by multi-angle laser light scattering and small-angle X-ray scattering in solution revealed that the enzyme adopted highly flexible unbound, latent structures and peptide-bound, active structures that differed from the AD13-MDTCS crystal structure. Moreover, the peptide behaved like a self-avoiding random chain. We integrated the results with computational approaches, derived an ensemble of structures that collectively satisfied all experimental restraints, and discussed the functional implications. The interaction conforms to a 'fuzzy complex' that follows a 'dynamic zipper' mechanism involving numerous reversible, weak but additive interactions that result in strong binding and cleavage. Our findings contribute to illuminating the biochemistry of the vWF:ADAMTS-13 axis., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

Retinoic acid receptors (RARs) and retinoid X receptors (RXRs) form heterodimers that activate target gene transcription by recruiting co-activator complexes in response to ligand binding. The nuclear receptor (NR) co-activator TIF2 mediates this recruitment by interacting with the ligand-binding domain (LBD) of NRs trough the nuclear receptor interaction domain (TIF2 NRID ) containing three highly conserved α-helical LxxLL motifs (NR-boxes). The precise binding mode of this domain to RXR/RAR is not clear due to the disordered nature of TIF2. Here we present the structural characterization of TIF2 NRID by integrating several experimental (NMR, SAXS, Far-UV CD, SEC-MALS) and computational data. Collectively, the data are in agreement with a largely disordered protein with partially structured regions, including the NR-boxes and their flanking regions, which are evolutionary conserved. NMR and X-ray crystallographic data on TIF2 NRID in complex with RXR/RAR reveal a multisite binding of the three NR-boxes as well as an active role of their flanking regions in the interaction., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

Linkers in polyproteins are considered as mere spacers between two adjacent domains. However, a series of studies using single-molecule force spectroscopy have recently reported distinct thermodynamic stability of I27 in polyproteins with varying linkers and indicated the vital role of linkers in domain stability. A flexible glycine rich linker (-(GGG) n , n ≥ 3) featured unfolding at lower forces than the regularly used arg-ser (RS) based linker. Interdomain interactions among I27 domains in Gly-rich linkers were suggested to lead to reduced domain stability. However, the negative impact of inter domain interactions on domain stability is thermodynamically counter-intuitive and demanded thorough investigations. Here, using an array of ensemble equilibrium experiments and in-silico measurements with I27 singlet and doublets with two aforementioned linkers, we delineate that the inter-domain interactions in fact raise the stability of the polyprotein with RS linker. More surprisingly, a highly flexible Gly-rich linker has no interference on the stability of polyprotein. Overall, we conclude that flexible linkers are preferred in a polyprotein for maintaining domain's independence., Competing Interests: Declaration of competing interest The authors declare no competing interest., (Copyright © 2021 Elsevier Inc. All rights reserved.)

Intrinsically disordered proteins (IDPs) play key functional roles facilitated by their inherent plasticity. In most of the cases, IDPs recognize their partners through partially structured elements inserted in fully disordered chains. The identification and characterization of these elements is fundamental to understand the functional mechanisms of IDPs. Although several computational methods have been developed to identify order within disordered chains, most of the current secondary structure predictors are focused on globular proteins and are not necessarily appropriate for IDPs. Here, we present a comprehensible method, called Local Structural Propensity Predictor (LS2P), to predict secondary structure elements from IDP sequences. LS2P performs statistical analyses from a database of three-residue fragments extracted from coil regions of high-resolution protein structures. In addition to identifying scarcely populated helical and extended regions, the method pinpoints short stretches triggering β-turn formation or promoting α-helices. The simplicity of the method enables a direct connection between experimental observations and structural features encoded in IDP sequences., Competing Interests: Declaration of Competing Interest The authors declare no conflict of interest., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

Clarification of solution structure and its modulation in proteins and protein complexes is crucially important to understand dynamical ordering in macromolecular systems. Small-angle x-ray scattering (SAXS) and small-angle neutron scattering (SANS) are among the most powerful techniques to derive structural information. Recent progress in sample preparation, instruments and software analysis is opening up a new era for small-angle scattering. In this review, recent progress and trends of SAXS and SANS are introduced from the point of view of instrumentation and analysis, touching on general features and standard methods of small-angle scattering. This article is part of a Special Issue entitled "Biophysical Exploration of Dynamical Ordering of Biomolecular Systems" edited by Dr. Koichi Kato., (Copyright © 2017. Published by Elsevier B.V.)

Nucleocapsid assembly is an essential process in the replication of the non-segmented, negative-sense RNA viruses (NNVs). Unassembled nucleoprotein (N(0)) is maintained in an RNA-free and monomeric form by its viral chaperone, the phosphoprotein (P), forming the N(0)-P complex. Our earlier work solved the structure of vesicular stomatitis virus complex formed between an N-terminally truncated N (NΔ21) and a peptide of P (P60) encompassing the N(0)-binding site, but how the full-length P interacts with N(0) remained unknown. Here, we combine several experimental biophysical methods including size exclusion chromatography with detection by light scattering and refractometry, small-angle X-ray and neutron scattering and nuclear magnetic resonance spectroscopy with molecular dynamics simulation and computational modeling to characterize the NΔ21(0)-PFL complex formed with dimeric full-length P. We show that for multi-molecular complexes, simultaneous multiple-curve fitting using small-angle neutron scattering data collected at varying contrast levels provides additional information and can help refine structural ensembles. We demonstrate that (a) vesicular stomatitis virus PFL conserves its high flexibility within the NΔ21(0)-PFL complex and interacts with NΔ21(0) only through its N-terminal extremity; (b) each protomer of P can chaperone one N(0) client protein, leading to the formation of complexes with stoichiometries 1N:P2 and 2N:P2; and (c) phosphorylation of residues Ser60, Thr62 and Ser64 provides no additional interactions with N(0) but creates a metal binding site in PNTR. A comparison with the structures of Nipah virus and Ebola virus N(0)-P core complex suggests a mechanism for the control of nucleocapsid assembly that is common to all NNVs., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

Intermediate conformations are crucial to our understanding of how proteins fold into their native structures and become functional. Conventional spectroscopic measurements of thermal denaturation transitions allow the detection of equilibrium intermediates but often provide little structural detail; thus, application of more informative techniques is required. Here we used small-angle X-ray scattering (SAXS) to study the thermal denaturation of four variants of Anabaena PCC 7119 flavodoxin, including the wild-type apo and holo forms, and two mutants, E20K/E72K and F98N. Denaturation was monitored from changes in SAXS descriptors. Although the starting and final points of the denaturation were similar for the flavodoxin variants tested, substantial differences in the unfolding pathway were apparent between them. In agreement with calorimetric data, analysis of the SAXS data sets indicated a three-state unfolding equilibrium for wild-type apoflavodoxin, a two-state equilibrium for the F98N mutant, and increased thermostability of the E20K/E72K mutant and holoflavodoxin. Although the apoflavodoxin intermediate consistently appeared mixed with significant amounts of either native or unfolded conformations, its SAXS profile was derived from the deconvolution of the temperature-dependent SAXS data set. The apoflavodoxin thermal intermediate was structurally close to the native state but less compact, thereby indicating incipient unfolding. The residues that foster denaturation were explored by an ensemble of equilibrium ϕ-value restrained molecular dynamics. These simulations pointed to residues located in the cofactor and partner-protein recognition regions as the initial sites of denaturation and suggest a conformational adaptation as the mechanism of action in apoflavodoxin., (Copyright © 2011 Elsevier Ltd. All rights reserved.)

X-ray crystallography and NMR can provide detailed structural information of protein-protein complexes, but technical problems make their application challenging in the high-throughput regime. Other methods such as small-angle X-ray scattering (SAXS) are more promising for large-scale application, but at the cost of lower resolution, which is a problem that can be solved by complementing SAXS data with theoretical simulations. Here, we propose a novel strategy that combines SAXS data and accurate protein-protein docking simulations. The approach has been benchmarked on a large pool of known structures with synthetic SAXS data, and on three experimental examples. The combined approach (pyDockSAXS) provided a significantly better success rate (43% for the top 10 predictions) than either of the two methods alone. Further analysis of the influence of different docking parameters made it possible to increase the success rates for specific cases, and to define guidelines for improving the data-driven protein-protein docking protocols., (Copyright © 2010 Elsevier Ltd. All rights reserved.)

Partly unfolded protein conformations close to the native state may play important roles in protein function and in protein misfolding. Structural analyses of such conformations which are essential for their fully physicochemical understanding are complicated by their characteristic low populations at equilibrium. We stabilize here with a single mutation the equilibrium intermediate of apoflavodoxin thermal unfolding and determine its solution structure by NMR. It consists of a large native region identical with that observed in the X-ray structure of the wild-type protein plus an unfolded region. Small-angle X-ray scattering analysis indicates that the calculated ensemble of structures is consistent with the actual degree of expansion of the intermediate. The unfolded region encompasses discontinuous sequence segments that cluster in the 3D structure of the native protein forming the FMN cofactor binding loops and the binding site of a variety of partner proteins. Analysis of the apoflavodoxin inner interfaces reveals that those becoming destabilized in the intermediate are more polar than other inner interfaces of the protein. Natively folded proteins contain hydrophobic cores formed by the packing of hydrophobic surfaces, while natively unfolded proteins are rich in polar residues. The structure of the apoflavodoxin thermal intermediate suggests that the regions of natively folded proteins that are easily responsive to thermal activation may contain cores of intermediate hydrophobicity., (Copyright (c) 2010 Elsevier Ltd. All rights reserved.)

The INhibitor of Growth (ING) family of tumor suppressors regulates the transcriptional state of chromatin by recruiting remodeling complexes to sites with histone H3 trimethylated at position K4 (H3K4me3). This modification is recognized by the plant homeodomain (PHD) present at the C-terminus in the five members of the ING family. ING4 facilitates histone H3 acetylation by the HBO1 complex. Here, we show that ING4 forms homodimers through its N-terminal domain, which folds independently into an elongated coiled-coil structure. The central region of ING4, which contains the nuclear localization sequence, is disordered and flexible and does not directly interact with p53, or does it with very low affinity, in contrast to previous findings. The NMR analysis of the full-length protein reveals that the two PHD fingers of the dimer are chemically equivalent and independent of the rest of the molecule. The detailed NMR analysis of the full-length dimeric protein binding to histone H3K4me3 shows essentially the same binding site and affinity as the isolated PHD finger. Therefore, the ING4 dimer has two identical and independent binding sites for H3K4me3 tails, which, in the context of the chromatin, could belong to the same or to different nucleosomes. These results show that ING4 is a bivalent reader of the chromatin H3K4me3 modification and suggest a mechanism for enhanced targeting of the HBO1 complex to specific chromatin sites. This mechanism could be common to other ING-containing remodeling complexes., ((c) 2009 Elsevier Ltd. All rights reserved.)

The N-terminal regions of the members of Src family of non-receptor protein tyrosine kinases are intrinsically unfolded and contain the maximum sequence divergence among them. In this study, we have addressed the structural characterization by nuclear magnetic resonance of this region of 84 residues that encompasses the SH4 and the unique domains (USrc) of the human c-Src. With this aim, the backbone assignment was performed using (13)C-detected experiments that overcome the spectral resolution problems and the large number of prolines that are typical for intrinsically unfolded proteins. The analysis of the residual dipolar couplings measured for the USrc indicates the presence of a low populated helical structure in the 60-75 region. No long-range contacts between remote fragments of the chain were detected with paramagnetic relaxation enhancement experiments. The structural characterization was extended to two different phosphorylation states of USrc that encompassed three different phosphorylated sites, Ser17, Thr37, and Ser75. The structural and conformational changes upon phosphorylation were monitored through chemical shift perturbations and residual dipolar couplings, indicating that modifications occur at local level and no global rearrangements were apparent. These results suggest a scenario where phosphorylation induces a global electrostatic perturbation that could be involved in the membrane unbinding of c-Src and that could be related with the localization of the enzyme. These observations suggest the unique domain of Src kinases as a source of selectivity and reinforce the relevant role of intrinsically disordered proteins in biological processes.

Src kinase plays an important role in several signaling and regulation mechanisms in vivo. Enzymatic activity is tightly regulated through the phosphorylation and dephosphorylation of tyrosine 527, which is placed at the C-terminal tail. Here, we have addressed domain rearrangements involved in the regulation mechanism of Src kinase in solution using small-angle X-ray scattering. In the phosphorylated wild-type form of Src kinase corresponding to the inactive state of the protein, a single conformation compatible with a closed crystallographic structure was found in solution. In the Y527F point mutant representing the active state, analysis of scattering data reveals an equilibrium between two differently populated conformations differing in the radius of gyration by 5 A. The major species (85% of the total population) presents a closed conformation indistinguishable from the crystallographic structure of the inactive state. The minor species (15% of the total population) is an open conformation similar to the crystallographic structure in the active state. The latter structure has the SH3, SH2, and SH2-catalytic domain linker assembled as a pseudo-two-domain protein. The regulation model emerging from this study, including at least three different conformational states, allows the tight regulation of the enzyme without compromising fast response in the presence of natural targets.

Residual dipolar couplings (RDCs) provide excellent probes for the exploration of dynamics in biomolecules on biologically relevant time-scales. Applying geometric motional models in combination with high-resolution structures to fit experimental RDCs allows the extraction of local dynamic amplitudes of peptide planes in proteins using only a limited number of data points. Here we compare the behaviour of three simple and intuitive dynamic modes: the Gaussian axial fluctuation model (1D-GAF), the two-site jump model, and a model supposing axially symmetric motion about a mean orientation. The requirement of a structural model makes this kind of methodology potentially very sensitive to structural imprecision. Numerical simulations of RDC dynamic averaging under different regimes show that the anisotropic motional models are more geometrically stringent than the axially symmetric model making it more difficult to alias structural noise as artificial dynamic amplitudes. Indeed, it appears that the model extracts accurate motional amplitudes even in the presence of significant structural error. We also show that a two-site jump model, also assuming the (alpha)C(i-1)-(alpha)C(i) as rotation axis, can only be distinguished from the previously developed GAF model beyond amplitude/jumps of around 40 degrees. The importance of appropriate estimation of the molecular alignment tensor for determination of local motional amplitudes is also illustrated here. We demonstrate a systematic scaling of extracted dynamic amplitudes if a static structure is assumed when determining the alignment tensor from dynamically averaged RDCs. As an example an artificial increase of 0.14 (0.85 compared to the expected 0.71) is observed in the extracted S2 if a pervasive 20 degrees GAF motion is present that is ignored in the tensor determination. Finally we apply a combined approach using the most appropriate motional model, to complete the analysis of dynamic motions from protein G.