Multivalent interactions of the disordered regions of XLF and XRCC4 foster robust cellular NHEJ and drive the formation of ligation-boosting condensates in vitro.

In mammalian cells, DNA double-strand breaks are predominantly repaired by non-homologous end joining (NHEJ). During repair, the Ku70/80 heterodimer (Ku), XRCC4 in complex with DNA Ligase 4 (X4L4), and XLF form a flexible scaffold that holds the broken DNA ends together. Insights into the architectural organization of the NHEJ scaffold and its regulation by the DNA-dependent protein kinase catalytic subunit (DNA-PKcs) have recently been obtained by single-particle cryo-electron microscopy analysis. However, several regions, especially the C-terminal regions (CTRs) of the XRCC4 and XLF scaffolding proteins, have largely remained unresolved in experimental structures, which hampers the understanding of their functions. Here, we used magnetic resonance techniques and biochemical assays to comprehensively characterize the interactions and dynamics of the XRCC4 and XLF CTRs at atomic resolution. We show that the CTRs of XRCC4 and XLF are intrinsically disordered and form a network of multivalent heterotypic and homotypic interactions that promotes robust cellular NHEJ activity. Importantly, we demonstrate that the multivalent interactions of these CTRs led to the formation of XLF and X4L4 condensates in vitro which can recruit relevant effectors and critically stimulate DNA end ligation. Our work highlights the role of disordered regions in the mechanism and dynamics of NHEJ and lays the groundwork for the investigation of NHEJ protein disorder and its associated condensates inside cells with implications in cancer biology, immunology and the development of genome editing strategies.

PP2A is activated by cytochrome c upon formation of a diffuse encounter complex with SET/TAF-Iß.

Intrinsic protein flexibility is of overwhelming relevance for intermolecular recognition and adaptability of highly dynamic ensemble of complexes, and the phenomenon is essential for the understanding of numerous biological processes. These conformational ensembles-encounter complexes-lack a unique organization, which prevents the determination of well-defined high resolution structures. This is the case for complexes involving the oncoprotein SET/template-activating factor-Iß (SET/TAF-Iß), a histone chaperone whose functions and interactions are significantly affected by its intrinsic structural plasticity. Besides its role in chromatin remodeling, SET/TAF-Iß is an inhibitor of protein phosphatase 2A (PP2A), which is a key phosphatase counteracting transcription and signaling events controlling the activity of DNA damage response (DDR) mediators. During DDR, SET/TAF-Iß is sequestered by cytochrome c (Cc) upon migration of the hemeprotein from mitochondria to the cell nucleus. Here, we report that the nuclear SET/TAF-Iß:Cc polyconformational ensemble is able to activate PP2A. In particular, the N-end folded, globular region of SET/TAF-Iß (a.k.a. SET/TAF-Iß ΔC)-which exhibits an unexpected, intrinsically highly dynamic behavior-is sufficient to be recognized by Cc in a diffuse encounter manner. Cc-mediated blocking of PP2A inhibition is deciphered using an integrated structural and computational approach, combining small-angle X-ray scattering, electron paramagnetic resonance, nuclear magnetic resonance, calorimetry and molecular dynamics simulations.

Nickel and GTP Modulate Helicobacter pylori UreG Structural Flexibility.

UreG is a P-loop GTP hydrolase involved in the maturation of nickel-containing urease, an essential enzyme found in plants, fungi, bacteria, and archaea. This protein couples the hydrolysis of GTP to the delivery of Ni(II) into the active site of apo-urease, interacting with other urease chaperones in a multi-protein complex necessary for enzyme activation. Whereas the conformation of Helicobacter pylori (Hp) UreG was solved by crystallography when it is in complex with two other chaperones, in solution the protein was found in a disordered and flexible form, defining it as an intrinsically disordered enzyme and indicating that the well-folded structure found in the crystal state does not fully reflect the behavior of the protein in solution. Here, isothermal titration calorimetry and site-directed spin labeling coupled to electron paramagnetic spectroscopy were successfully combined to investigate HpUreG structural dynamics in solution and the effect of Ni(II) and GTP on protein mobility. The results demonstrate that, although the protein maintains a flexible behavior in the metal and nucleotide bound forms, concomitant addition of Ni(II) and GTP exerts a structural change through the crosstalk of different protein regions.

The Hunt for the Closed Conformation of the Fruit-Ripening Enzyme 1-Aminocyclopropane-1-carboxylic Oxidase: A Combined Electron Paramagnetic Resonance and Molecular Dynamics Study.

1-Aminocyclopropane-1-carboxylic oxidase (ACCO) is a non-heme iron(II)-containing enzyme involved in the biosynthesis of the phytohormone ethylene, which regulates fruit ripening and flowering in plants. The active conformation of ACCO, and in particular that of the C-terminal part, remains unclear and open and closed conformations have been proposed. In this work, a combined experimental and computational study to understand the conformation and dynamics of the C-terminal part is reported. Site-directed spin-labeling coupled to electron paramagnetic resonance (SDSL-EPR) spectroscopy was used. Mutagenesis experiments were performed to generate active enzymes bearing two paramagnetic labels (nitroxide radicals) anchored on cysteine residues, one in the main core and one in the C-terminal part. Inter-spin distance distributions were measured by pulsed EPR spectroscopy and compared with the results of molecular dynamics simulations. The results reveal the existence of a flexibility of the C-terminal part. This flexibility generates several conformations of the C-terminal part of ACCO that correspond neither to the existing crystal structures nor to the modelled structures. This highly dynamic region of ACCO raises questions on its exact function during enzymatic activity.

Chemical Modification of 1-Aminocyclopropane Carboxylic Acid (ACC) Oxidase: Cysteine Mutational Analysis, Characterization, and Bioconjugation with a Nitroxide Spin Label.

1-Aminocyclopropane carboxylic acid oxidase (ACCO) catalyzes the last step of ethylene biosynthesis in plants. Although some sets of structures have been described, there are remaining questions on the active conformation of ACCO and in particular, on the conformation and potential flexibility of the C-terminal part of the enzyme. Several techniques based on the introduction of a probe through chemical modification of amino acid residues have been developed for determining the conformation and dynamics of proteins. Cysteine residues are recognized as convenient targets for selective chemical modification of proteins, thanks to their relatively low abundance in protein sequences and to their well-mastered chemical reactivity. ACCOs have generally 3 or 4 cysteine residues in their sequences. By a combination of approaches including directed mutagenesis, activity screening on cell extracts, biophysical and biochemical characterization of purified enzymes, we evaluated the effect of native cysteine replacement and that of insertion of cysteines on the C-terminal part in tomato ACCO. Moreover, we have chosen to use paramagnetic labels targeting cysteine residues to monitor potential conformational changes by electron paramagnetic resonance (EPR). Given the level of conservation of the cysteines in ACCO from different plants, this work provides an essential basis for the use of cysteine as probe-anchoring residues.

Author Correction: Iron restriction inside macrophages regulates pulmonary host defense against Rhizopus species.

The original version of this Article contained an error in the spelling of the author Emilien Etienne, which was incorrectly given as Emilien Ettiene. These errors have now been corrected in both the PDF and HTML versions of the Article.

Two Tau binding sites on tubulin revealed by thiol-disulfide exchanges.

Tau is a Microtubule-associated protein that induces and stabilizes the formation of the Microtubule cytoskeleton and plays an important role in neurodegenerative diseases. The Microtubules binding region of Tau has been determined for a long time but where and how Tau binds to its partner still remain a topic of debate. We used Site Directed Spin Labeling combined with EPR spectroscopy to monitor Tau upon binding to either Taxol-stabilized MTs or to αß-tubulin when Tau is directly used as an inducer of MTs formation. Using maleimide-functionalized labels grafted on the two natural cysteine residues of Tau, we found in both cases that Tau remains highly flexible in these regions confirming the fuzziness of Tau:MTs complexes. More interestingly, using labels linked by a disulfide bridge, we evidenced for the first time thiol disulfide exchanges between αß-tubulin or MTs and Tau. Additionally, Tau fragments having the two natural cysteines or variants containing only one of them were used to determine the role of each cysteine individually. The difference observed in the label release kinetics between preformed MTs or Tau-induced MTs, associated to a comparison of structural data, led us to propose two putative binding sites of Tau on αß-tubulin.

Iron restriction inside macrophages regulates pulmonary host defense against Rhizopus species.

Mucormycosis is a life-threatening respiratory fungal infection predominantly caused by Rhizopus species. Mucormycosis has incompletely understood pathogenesis, particularly how abnormalities in iron metabolism compromise immune responses. Here we show how, as opposed to other filamentous fungi, Rhizopus spp. establish intracellular persistence inside alveolar macrophages (AMs). Mechanistically, lack of intracellular swelling of Rhizopus conidia results in surface retention of melanin, which induces phagosome maturation arrest through inhibition of LC3-associated phagocytosis. Intracellular inhibition of Rhizopus is an important effector mechanism, as infection of immunocompetent mice with swollen conidia, which evade phagocytosis, results in acute lethality. Concordantly, AM depletion markedly increases susceptibility to mucormycosis. Host and pathogen transcriptomics, iron supplementation studies, and genetic manipulation of iron assimilation of fungal pathways demonstrate that iron restriction inside macrophages regulates immunity against Rhizopus. Our findings shed light on the pathogenetic mechanisms of mucormycosis and reveal the role of macrophage-mediated nutritional immunity against filamentous fungi.

Orthogonal Tyrosine and Cysteine Site-Directed Spin Labeling for Dipolar Pulse EPR Spectroscopy on Proteins.

Site-directed spin labeling of native tyrosine residues in isolated domains of the protein PTBP1, using a Mannich-type reaction, was combined with conventional spin labeling of cysteine residues. Double electron-electron resonance (DEER) EPR measurements were performed for both the nitroxide-nitroxide and Gd(III)-nitroxide label combinations within the same protein molecule. For the prediction of distance distributions from a structure model, rotamer libraries were generated for the two linker forms of the tyrosine-reactive isoindoline-based nitroxide radical Nox. Only moderate differences exist between the spatial spin distributions for the two linker forms of Nox. This strongly simplifies DEER data analysis, in particular, if only mean distances need to be predicted.

Dynamics of the intrinsically disordered protein CP12 in its association with GAPDH in the green alga Chlamydomonas reinhardtii: a fuzzy complex.

CP12 is a widespread regulatory protein of oxygenic photosynthetic organisms that contributes to the regulation of the Calvin cycle by forming a supra-molecular complex with at least two enzymes: glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and phosphoribulokinase (PRK). CP12 shares some similarities with intrinsically disordered proteins (IDPs) depending on its redox state. In this study, site-directed spin labeling (SDSL) combined with EPR spectroscopy was used to probe the dynamic behavior of CP12 from Chlamydomonas reinhardtii upon binding to GAPDH, the first step towards ternary complex formation. The two N-terminal cysteine residues were labeled using the classical approach while the tyrosine located at the C-terminal end of CP12 was modified following an original procedure. The results show that the label grafted at the C-terminal extremity is in the vicinity of the interaction site whereas the N-terminal region remains fully disordered upon binding to GAPDH. In conclusion, GAPDH-CP12 is a fuzzy complex, in which the N-terminal region of CP12 keeps a conformational freedom in the bound form. This fuzziness could be one of the keys to facilitate binding of PRK to CP12-GAPDH and to form the ternary supra-molecular complex.

Enlarging the panoply of site-directed spin labeling electron paramagnetic resonance (SDSL-EPR): sensitive and selective spin-labeling of tyrosine using an isoindoline-based nitroxide.

Site-directed spin labeling (SDSL) combined with electron paramagnetic resonance (EPR) spectroscopy has emerged as a powerful approach to study structure and dynamics in proteins. One limitation of this approach is the fact that classical spin labels are functionalized to be grafted on natural or site-directed mutagenesis generated cysteine residues. Despite the widespread success of cysteine-based modification strategies, the technique becomes unsuitable when cysteine residues play a functional or structural role in the protein under study. To overcome this limitation, we propose an isoindoline-based nitroxide to selectively target tyrosine residues using a Mannich type reaction, the feasibility of which has been demonstrated in a previous study. This nitroxide has been synthesized and successfully grafted successively on p-cresol, a small tetrapeptide and a model protein: a small chloroplastic protein CP12 having functional cysteines and a single tyrosine. Studying the association of the labeled CP12 with its partner protein, we showed that the isoindoline-based nitroxide is a good reporter to reveal changes in its local environment contrary to the previous study where the label was poorly sensitive to probe structural changes. The successful targeting of tyrosine residues with the isoindoline-based nitroxide thus offers a highly promising approach, complementary to the classical cysteine-SDSL one, which significantly enlarges the field of applications of the technique for probing protein dynamics.

Monitoring structural transitions in IDPs by site-directed spin labeling EPR spectroscopy.

Electron paramagnetic resonance (EPR) spectroscopy is a technique that specifically detects unpaired electrons. EPR sensitive reporter groups (spin labels or spin probes) can be introduced into biological systems via site-directed spin labeling (SDSL). This is usually accomplished by cysteine-substitution mutagenesis followed by covalent modification of the unique sulfhydryl group with a selective nitroxide reagent. SDSL EPR spectroscopy has been shown to be a sensitive and powerful method to study structural transitions within intrinsically disordered proteins (IDPs). In this chapter, we provide a detailed experimental protocol for this approach and present a few examples of EPR spectral shapes illustrative of various mobility regimes of the spin probe, reflecting different protein topologies.

Probing structural transitions in both structured and disordered proteins using site-directed spin-labeling EPR spectroscopy.

EPR spectroscopy is a technique that specifically detects unpaired electrons. EPR-sensitive reporter groups (spin labels or spin probes) can be introduced into biological systems via site-directed spin-labeling (SDSL). The basic strategy of SDSL involves the introduction of a paramagnetic group at a selected protein site. This is usually accomplished by cysteine-substitution mutagenesis, followed by covalent modification of the unique sulfydryl group with a selective reagent bearing a nitroxide radical. In this review we briefly describe the theoretical principles of this well-established approach and illustrate how we successfully applied it to investigate structural transitions in both human pancreatic lipase (HPL), a protein with a well-defined α/ß hydrolase fold, and the intrinsically disordered C-terminal domain of the measles virus nucleoprotein (N(TAIL) ) upon addition of ligands and/or protein partners. In both cases, SDSL EPR spectroscopy allowed us to document protein conformational changes at the residue level. The studies herein summarized show that this approach is not only particularly well-suited to study IDPs that inherently escape atomistic description by X-ray crystallography but also provides dynamic information on structural transitions occurring within well-characterized structured proteins for which X-ray crystallography can only provide snapshots of the initial and final stages.

Conformational analysis of the partially disordered measles virus N(TAIL)-XD complex by SDSL EPR spectroscopy.

To characterize the structure of dynamic protein systems, such as partly disordered protein complexes, we propose a novel approach that relies on a combination of site-directed spin-labeled electron paramagnetic resonance spectroscopy and modeling of local rotation conformational spaces. We applied this approach to the intrinsically disordered C-terminal domain of the measles virus nucleoprotein (N(TAIL)) both free and in complex with the X domain (XD, aa 459-507) of the viral phosphoprotein. By comparing measured and modeled temperature-dependent restrictions of the side-chain conformational spaces of 12 SL cysteine-substituted N(TAIL) variants, we showed that the 490-500 region of N(TAIL) is prestructured in the absence of the partner, and were able to quantitatively estimate, for the first time to our knowledge, the extent of the alpha-helical sampling of the free form. In addition, we showed that the 505-525 region of N(TAIL) conserves a significant degree of freedom even in the bound form. The latter two findings provide a mechanistic explanation for the reported rather high affinity of the N(TAIL)-XD binding reaction. Due to the nanosecond timescale of X-band EPR spectroscopy, we were also able to monitor the disordering in the 488-525 region of N(TAIL), in particular the unfolding of the alpha-helical region when the temperature was increased from 281 K to 310 K.

Amplitude of pancreatic lipase lid opening in solution and identification of spin label conformational subensembles by combining continuous wave and pulsed EPR spectroscopy and molecular dynamics.

The opening of the lid that controls the access to the active site of human pancreatic lipase (HPL) was measured from the magnetic interaction between two spin labels grafted on this enzyme. One spin label was introduced at a rigid position in HPL where an accessible cysteine residue (C181) naturally occurs. A second spin label was covalently bound to the mobile lid after introducing a cysteine residue at position 249 by site-directed mutagenesis. Double electron-electron resonance (DEER) experiments allowed the estimation of a distance of 19 +/- 2 A between the spin labels when bilabeled HPL was alone in a frozen solution, i.e., with the lid in the closed conformation. A magnetic interaction was however detected by continuous wave EPR experiments, suggesting that a fraction of bilabeled HPL contained spin labels separated by a shorter distance. These results could be interpreted by the presence of two conformational subensembles for the spin label lateral chain at position 249 when the lid was closed. The existence of these conformational subensembles was revealed by molecular dynamics experiments and confirmed by the simulation of the EPR spectrum. When the lid opening was induced by the addition of bile salts and colipase, a larger distance of 43 +/- 2 A between the two spin labels was estimated from DEER experiments. The distances measured between the spin labels grafted at positions 181 and 249 were in good agreement with those estimated from the known X-ray structures of HPL in the closed and open conformations, but for the first time, the amplitude of the lid opening was measured in solution or in a frozen solution in the presence of amphiphiles.

A new function of GAPDH from Chlamydomonas reinhardtii: a thiol-disulfide exchange reaction with CP12.

CP12 is a flexible protein that is well-known to interact with GAPDH, and this association is crucial to the regulation of enzyme activity. This regulation is likely related to structural transitions of both proteins, but the molecular bases of these changes are not yet understood. To answer this issue, we undertook a study based on the use of paramagnetic probes grafted on cysteine residues and followed by EPR spectroscopy. We present a new application of this approach that enables us to probe the functional role of cysteine residues in protein-protein interactions. Algal CP12 contains four cysteine residues involved in two disulfide bridges in its oxidized state and has some alpha-helical secondary structural elements. In contrast, in its reduced state, CP12 is mainly unstructured and shares some physical properties with intrinsically disordered proteins. Treatment of CP12 with a methane thiosulfonate derivative spin-label (MTSL) led to the labeling of the cysteine residues involved in the C-terminal bridge only as revealed by mass spectrometry. Surprisingly, the partner protein GAPDH induced the cleavage of the disulfide bridge between the cysteine residues of CP12 and the spin-label, resulting in the full release of the label. We showed the existence of a transitory interaction between both proteins and proposed a mechanism based on a thiol-disulfide exchange reaction. The results of this study point out a novel role of the algal GAPDH which is often termed a "moonlighting" protein.

Mapping alpha-helical induced folding within the intrinsically disordered C-terminal domain of the measles virus nucleoprotein by site-directed spin-labeling EPR spectroscopy.

Using site-directed spin-labeling EPR spectroscopy, we mapped the region of the intrinsically disordered C-terminal domain of measles virus nucleoprotein (N(TAIL)) that undergoes induced folding. In addition to four spin-labeled N(TAIL) variants (S407C, S488C, L496C, and V517C) (Morin et al. (2006), J Phys Chem 110: 20596-20608), 10 new single-site cysteine variants were designed, purified from E. coli, and spin-labeled. These 14 spin-labeled variants enabled us to map in detail the gain of rigidity of N(TAIL) in the presence of either the secondary structure stabilizer 2,2,2-trifluoroethanol or the C-terminal domain X (XD) of the viral phosphoprotein. Different regions of N(TAIL) were shown to contribute to a different extent to the binding to XD, while the mobility of the spin labels grafted at positions 407 and 460 was unaffected upon addition of XD; that of the spin labels grafted within the 488-502 and the 505-522 regions was severely and moderately reduced, respectively. Furthermore, EPR experiments in the presence of 30% sucrose allowed us to precisely map to residues 488-502, the N(TAIL) region undergoing alpha-helical folding. The mobility of the 488-502 region was found to be restrained even in the absence of the partner, a behavior that could be accounted for by the existence of a transiently populated folded state. Finally, we show that the restrained motion of the 505-522 region upon binding to XD is due to the alpha-helical transition occurring within the 488-502 region and not to a direct interaction with XD.

Changing the ligation of the distal [4Fe4S] cluster in NiFe hydrogenase impairs inter- and intramolecular electron transfers.

In NiFe hydrogenases, electrons are transferred from the active site to the redox partner via a chain of three Iron-Sulfur clusters, and the surface-exposed [4Fe4S] cluster has an unusual His(Cys)3 ligation. When this Histidine (H184 in Desulfovibrio fructosovorans) is changed into a cysteine or a glycine, a distal cubane is still assembled but the oxidative activity of the mutants is only 1.5 and 3% of that of the WT, respectively. We compared the activities of the WT and engineered enzymes for H2 oxidation, H+ reduction and H/D exchange, under various conditions: (i) either with the enzyme directly adsorbed onto an electrode or using soluble redox partners, and (ii) in the presence of exogenous ligands whose binding to the exposed Fe of H184G was expected to modulate the properties of the distal cluster. Protein film voltammetry proved particularly useful to unravel the effects of the mutations on inter and intramolecular electron transfer (ET). We demonstrate that changing the coordination of the distal cluster has no effect on cluster assembly, protein stability, active-site chemistry and proton transfer; however, it slows down the first-order rates of ET to and from the cluster. All-sulfur coordination is actually detrimental to ET, and intramolecular (uphill) ET is rate determining in the glycine variant. This demonstrates that although [4Fe4S] clusters are robust chemical constructs, the direct protein ligands play an essential role in imparting their ability to transfer electrons.