Coordinated Rearrangements between Cytoplasmic and Periplasmic Domains of the Membrane Protein Complex ExbB-ExbD of Escherichia coli

SummaryGram-negative bacteria rely on the ExbB-ExbD-TonB system for the import of essential nutrients. Despite decades of research, the stoichiometry, subunit organization, and mechanism of action of the membrane proteins of the Ton system remain unclear. We copurified ExbB with ExbD as an ∼240 kDa protein-detergent complex, measured by light scattering and by native gels. Quantitative Coomassie staining revealed a stoichiometry of ExbB4-ExbD2. Negative stain electron microscopy and 2D analysis showed particles of ∼10 nm diameter in multiple structural states. Nanogold labeling identified the position of the ExbD periplasmic domain. Random conical tilt was used to reconstruct the particles in three structural states followed by sorting of the single particles and refinement of each state. The different states are interpreted by coordinated structural rearrangements between the cytoplasmic domain and the periplasmic domain, concordant with in vivo predictions

An atypical BRCT-BRCT interaction with the XRCC1 scaffold protein compacts human DNA ligase IIIα within a flexible DNA repair complex

The XRCC1-DNA ligase IIIα complex (XL) is critical for DNA single-strand break repair, a key target for PARP inhibitors in cancer cells deficient in homologous recombination. Here, we combined biophysical approaches to gain insights into the shape and conformational flexibility of the XL as well as XRCC1 and DNA ligase IIIα (LigIIIα) alone. Structurally-guided mutational analyses based on the crystal structure of the human BRCT-BRCT heterodimer identified the network of salt bridges that together with the N-terminal extension of the XRCC1 C-terminal BRCT domain constitute the XL molecular interface. Coupling size exclusion chromatography with small angle X-ray scattering and multiangle light scattering (SEC-SAXS-MALS), we determined that the XL is more compact than either XRCC1 or LigIIIα, both of which form transient homodimers and are highly disordered. The reduced disorder and flexibility allowed us to build models of XL particles visualized by negative stain electron microscopy that predict close spatial organization between the LigIIIα catalytic core and both BRCT domains of XRCC1. Together our results identify an atypical BRCT-BRCT interaction as the stable nucleating core of the XL that links the flexible nick sensing and catalytic domains of LigIIIα to other protein partners of the flexible XRCC1 scaffold

AI is a viable alternative to high throughput screening: a 318-target study

: High throughput screening (HTS) is routinely used to identify bioactive small molecules. This requires physical compounds, which limits coverage of accessible chemical space. Computational approaches combined with vast on-demand chemical libraries can access far greater chemical space, provided that the predictive accuracy is sufficient to identify useful molecules. Through the largest and most diverse virtual HTS campaign reported to date, comprising 318 individual projects, we demonstrate that our AtomNet® convolutional neural network successfully finds novel hits across every major therapeutic area and protein class. We address historical limitations of computational screening by demonstrating success for target proteins without known binders, high-quality X-ray crystal structures, or manual cherry-picking of compounds. We show that the molecules selected by the AtomNet® model are novel drug-like scaffolds rather than minor modifications to known bioactive compounds. Our empirical results suggest that computational methods can substantially replace HTS as the first step of small-molecule drug discovery

Energy-transducing proteins of «Escherichia coli»: lipid-based crystallization trials

Gram-negative bacteria such as Escherichia coli possess a cell envelope composed of an outer membrane (OM) and a cytoplasmic membrane (CM). Widespread antibiotic resistance among these bacteria has necessitated the elucidation of novel therapeutic targets. Scarce nutrients such as iron and vitamin B12 cross the OM by energy-dependent transport. Since the OM lacks a source of energy, bacteria couple the energized CM to drive this process. The TonB-ExbB-ExbD integral membrane protein complex in the CM harnesses the proton gradient and transduces this energy to OM receptors. Despite decades of informative genetic and biochemical research, limited structural and functional information is available on this complex. In particular, only partial structures of the periplasmic domains of TonB and ExbD are known and all structural information is lacking for ExbB, considered to be the scaffold protein of the complex. Furthermore, the stoichiometry of the complex and the putative proton translocation pathway remain controversial. To gain insight into the molecular mechanisms of bacterial scarce nutrient transport, we produced, purified and characterized the ExbB and ExbD proteins before initiating lipid-based crystallization trials. The proteins were produced in milligram amounts and purified as a complex. The complex was found to be ~400 kDa and monodisperse by analytical size exclusion chromatography (SEC). Nuclear magnetic resonance-based detergent quantitation identified excess detergent that concentrated as a function of protein concentration. This led to greater apparent molecular weight of the complex by SEC. The excess detergent was able to be diminished by adsorbent beads or exchanged to a related, dialyzable detergent. Bicelle and in meso crystallization trials were initiated with purified ExbB-ExbD complexes, with promising leads identified. Optimization is ongoing to develop X-ray diffraction-quality crystals of ExbB-ExbD. These preliminary successes have formed the basis for the pursuit of ExbB-ExbD complex structure determination by lipid-based crystallization, a key to understand scarce nutrient import in Gram-negative bacteria.L'identification de nouvelles cibles thérapeutiques est rendue nécessaire par la fréquence des cas de résistance aux agents antimicrobiens chez les bactéries. Les bactéries à Gram négatif tel Escherichia coli possèdent une enveloppe cellulaire constituée d'une membrane externe (ME) et d'une membrane cytoplasmique (MC). Par ailleurs, les nutriments rares tel le fer et la vitamine B12 doivent traverser la ME par un transport exigeant de l'énergie. Cependant, puisque la ME est dénuée d'une source d'énergie, les bactéries doivent la coupler à la MC énergisée pour alimenter ces processus de transport. On sait déjà que le complexe protéique membranaire TonB-ExbB-ExbD, situé à la MC, harnache la force proton-motrice et achemine l'énergie aux récepteurs de la ME. Malgré des décennies de recherche sur la génétique et la biochimie de ce complexe, on ne dispose que d'informations limitées sur sa structure et sa fonction. En particulier, seules les structures des domaines périplasmiques de TonB et ExbD sont connues et aucune information structurale n'est disponible pour ExbB, dont le rôle de protéine d'échafaudage est connu. Qui plus est, la stoechiométrie du complexe et le chemin supposé des protons à travers la MC restent sujets à controverse. Afin de mieux connaître les mécanismes moléculaires du transport des nutriments rares chez les bactéries à Gram négatif, nous avons produit, purifié et charactérisé les protéines ExbB et ExbD et avons démarré des essais de cristallisation en phase lipidique. Nous arrivons à produire plusieurs milligrammes de ces protéines sous la forme d'un complexe. Par filtration sur gel (FG), nous avons évalué le poids moléculaire du complexe, qui est monodispersé, à ~400 kDa. En outre, nous avons établi par résonance magnétique nucléaire que nos échantillons d'ExbB-ExbD contenaient un excès de détergent et que la concentration de ce dernier et celle du complexe étaient corrélées. De ce fait, la taille du complexe parait plus élevée par FG. Nous avons diminué le surplus de détergent grâce à des billes adsorbantes ou en échangeant le détergent pour un amphiphile similaire mais dialysable. Nos essais de cristallisation d'ExbB-ExbD in meso et en bicelles ont identifié des pistes prometteuses. Des efforts d'optimisation sont en cours pour développer des cristaux d'ExbB-ExbD d'une qualité permettant la diffraction des rayons X. Ces réussites préliminaires permettent de poursuivre les efforts de cristallisation du complexe ExbB-ExbD en phase lipidique et ouvrent la voie à la résolution de sa structure. Une telle structure serait une contribution importante à notre compréhension du transport des nutriments rares chez les bactéries Gram-négatives

Stoichiometry and structures of ExbB-ExbD and ExbB-ExbD-TonB membrane protein complexes

Iron occupies a central position in almost all known life due to its abundance and chemistry. However, its catalytic nature can be dangerous if left unregulated. Iron surplus is a virulence factor during bacterial infections if not adequately sequestered in the host. Among other mechanisms, bacteria acquire iron using siderophores. Whereas Gram-positive bacteria transport heme and siderophores from their cognate surface receptors to importers at the cytoplasmic membrane (CM), Gram-negative bacteria must energize substrate translocation across the outer membrane. Energy at the CM is harnessed by the membrane proteins ExbB and ExbD and transduced to TonB-dependent transporters (TBDTs) by TonB. Apart from the C-terminal domains of TonB and ExbD, no structural information existed for the full-length proteins or for ExbB. While the three proteins assemble within a complex, the stoichiometry has remained elusive until now. Although distinct roles for ExbB and ExbD have been proposed, their structural arrangement with TonB and rearrangements throughout its work-cycle have hitherto not been demonstrated.To answer some of the fundamental questions about the Ton system, we undertook the co-purification of the complex formed by ExbB and ExbD. Following two chromatographic steps, we isolated the ~240 kDa detergent-solubilized ExbB4–ExbD2 complex. Uniform particles of ~10 nm diameter were observed by negative stain electron microscopy (EM). Although the complex was biochemically homogeneous, the particles displayed apparent conformational heterogeneity. Nanogold labeling oriented the soluble domains of ExbD and ExbB on either side of the detergent micelle. Three-dimensional (3D) reconstruction of the three principal conformations allowed us to propose a model whereby periplasmic ExbD homodimerization coordinates with cytoplasmic rearrangement of ExbB. Our reported stoichiometry and structural observations of cytoplasmic-periplasmic domain communication are consistent with in vivo predictions.The solubilising detergent of the ExbB4–ExbD2 complex was replaced by amphipols. Amphipols permitted us to utilize techniques normally reserved for soluble proteins, leading to our proposal for the arrangement of the transmembrane (TM) domains of the ExbB4–ExbD2 and ExbB6–ExbD4 complexes. Finally, small-angle scattering was used as an independent criterion to confirm the EM-derived size and shape of the ExbB4–ExbD2 complex.To gain insight into how ExbB and ExbD assemble with TonB, we co-purified the three proteins and determined their stoichiometry to be ExbB4–ExbD1–TonB1. Using phage display, we predicted sites of interaction between ExbD with itself and with TonB. The two- and three-protein complexes were similar in apparent size and detergent content, but differed in electronegativity. The ExbB4–ExbD1–TonB1 complex resembled the two-protein complex when observed by EM. Significantly, ExbD and TonB displayed two forms of interactions between their periplasmic domains: distal and extensive heterodimerization. 3D reconstruction of three representative conformations shed light on in vivo cross-linking and proteolytic studies.Combining all data into a model, we propose that ExbD positions TonB by extensive dimerization for contacting TBDTs. Distal dimerization would represent the energy-competent form of ExbD with TonB. Once bound to the Ton box, TonB would transmit a signal through its TM domain to ExbB. ExbB would then undergo a rearrangement and mediate ExbD-bound proton translocation into the cytoplasm. The energy transduction to TBDTs would involve the purported mutarotation of TonB and the unzipping of the TBDT β-sheet plug domain. A unique system responsible for energizing all siderophore import in Gram-negative bacteria is an attractive target for therapeutic development. Further structural determination by the complementary techniques of cryo-EM and crystallography hold promise in unraveling the mechanism of action of the Ton system.De par son abondance et sa chimie, le fer occupe une position centrale pour la plupart des formes de vie. Ses propriétés catalytiques le rendent cependant dangereux s’il n’est pas régulé. Alors que les bactéries à Gram positif transportent l’hème et les sidérophores à partir de récepteurs à la surface vers des transporteurs à la membrane cytoplasmique (MC), les bactéries à Gram négatif doivent utiliser de l’énergie pour transloquer ces substrats au travers de leur membrane externe. L’énergie à la MC est exploitée par les protéines membranaires ExbB et ExbD et transduite par TonB vers des transporteurs TonB dépendants (TTBDs). Alors que nous savons que ces trois protéines s’assemblent en un complexe, la stœchiométrie de celui-ci demeurait incomprise. De plus, même s’il fût suggéré qu’ExbB et ExbD ont des rôles distincts, leurs arrangement et réarrangements structurels avec TonB au cours du cycle de translocation n’avaient pas encore été élucidés.Afin de répondre à certaines des questions fondamentales sur le système Ton, nous avons procédé à la co-purification des complexes formés par ExbB et ExbD. Après deux rondes de purification par chromatographie, nous sommes parvenus à isoler un complexe d’environ 240 kDa, soluble dans un détergent d’ExbB4–ExbD2. Des particules uniformes d’un diamètre d’environ 10 nm ont par la suite pu être observées par microscopie électronique (ME) à coloration négative. Une reconstitution tridimensionnelle des trois principales conformations observées nous permet de proposer un modèle où l’homodimérization périplasmique d’ExbD coordonne avec le réarrangement cytoplasmique d’ExbB. De plus, la stœchiométrie et les observations structurelles de communication des domaines périplasmiques et cytoplasmiques que nous avons observées sont conformes aux prédictions formulées par les observations in vivo.Afin de mieux comprendre comment ExbB et ExbD s’assemblent avec TonB, nous avons co-purifié les trois protéines et avons déterminé que leur stœchiométrie devait être ExbB4–ExbD1–TonB1. En utilisant le phage display, nous avons pu proposer l’existence de sites d’interactions entre la protéine ExbD et elle-même ainsi qu’avec TonB dans le périplasme. Les complexes de deux ou trois protéines avaient une apparence, une taille et possédaient une quantité de détergents similaires, mais ils possédaient des propriétés électronégatives différentes. Le complexe ExbB4–ExbD1–TonB1 ressemblait au complexe de deux protéines lorsqu’observé par ME. Il est important de noter qu’ExbD et TonB affichaient deux types d’interaction entre leurs domaines périplasmiques : une interaction distale ainsi qu’une hétérodimérisation étendue. La reconstruction tridimensionnelle des trois conformations représentatives obtenues permet de mieux comprendre et interpréter les études de protéolyses et de réticulations effectuées in vivo.En combinant toutes ces données en un modèle, nous proposons qu’ExbD utilise une importante dimérisation pour positionner TonB afin de lui permettre d’entrer en contact avec les TTBDs. La dimérisation distale représenterait la forme capable d’utiliser de l’énergie d’ExbD et TonB. Une fois liée à la boite Ton, TonB peut transmettre un signal à travers son domaine transmembranaire vers ExbB. ExbB subit alors un réarrangement qui assure la translocation du proton lié à ExbD vers le cytoplasme. La transduction d’énergie vers les TTBDs impliquerait la mutarotation de TonB et la décontraction du feuillet β présent dans le domaine d’ancrage des TTBDs. La présence d’un système unique responsable de la conversion d’énergie nécessaire à l’importation de tous les sidérophores chez les bactéries à Gram négatif constituerait une cible attrayante de développement thérapeutique. Des études structurelles utilisant des techniques complémentaires comme la cryo-ME et la cristallographie semblent être une approche prometteuse afin de démystifier les mécanismes d’action du système Ton

Direct interaction of DNA repair protein tyrosyl DNA phosphodiesterase 1 and the DNA ligase III catalytic domain is regulated by phosphorylation of its flexible N-terminus.

Tyrosyl DNA phosphodiesterase 1 (TDP1) and DNA Ligase IIIα (LigIIIα) are key enzymes in single-strand break (SSB) repair. TDP1 removes 3'-tyrosine residues remaining after degradation of DNA topoisomerase (TOP) 1 cleavage complexes trapped by either DNA lesions or TOP1 inhibitors. It is not known how TDP1 is linked to subsequent processing and LigIIIα-catalyzed joining of the SSB. Here we define a direct interaction between the TDP1 catalytic domain and the LigIII DNA-binding domain (DBD) regulated by conformational changes in the unstructured TDP1 N-terminal region induced by phosphorylation and/or alterations in amino acid sequence. Full-length and N-terminally truncated TDP1 are more effective at correcting SSB repair defects in TDP1 null cells compared with full-length TDP1 with amino acid substitutions of an N-terminal serine residue phosphorylated in response to DNA damage. TDP1 forms a stable complex with LigIII170-755, as well as full-length LigIIIα alone or in complex with the DNA repair scaffold protein XRCC1. Small-angle X-ray scattering and negative stain electron microscopy combined with mapping of the interacting regions identified a TDP1/LigIIIα compact dimer of heterodimers in which the two LigIII catalytic cores are positioned in the center, whereas the two TDP1 molecules are located at the edges of the core complex flanked by highly flexible regions that can interact with other repair proteins and SSBs. As TDP1and LigIIIα together repair adducts caused by TOP1 cancer chemotherapy inhibitors, the defined interaction architecture and regulation of this enzyme complex provide insights into a key repair pathway in nonmalignant and cancer cells

Direct interaction of DNA repair protein tyrosyl DNA phosphodiesterase 1 and the DNA ligase III catalytic domain is regulated by phosphorylation of its flexible N-terminus.

Tyrosyl DNA phosphodiesterase 1 (TDP1) and DNA Ligase IIIα (LigIIIα) are key enzymes in single-strand break (SSB) repair. TDP1 removes 3'-tyrosine residues remaining after degradation of DNA topoisomerase (TOP) 1 cleavage complexes trapped by either DNA lesions or TOP1 inhibitors. It is not known how TDP1 is linked to subsequent processing and LigIIIα-catalyzed joining of the SSB. Here we define a direct interaction between the TDP1 catalytic domain and the LigIII DNA-binding domain (DBD) regulated by conformational changes in the unstructured TDP1 N-terminal region induced by phosphorylation and/or alterations in amino acid sequence. Full-length and N-terminally truncated TDP1 are more effective at correcting SSB repair defects in TDP1 null cells compared with full-length TDP1 with amino acid substitutions of an N-terminal serine residue phosphorylated in response to DNA damage. TDP1 forms a stable complex with LigIII170-755, as well as full-length LigIIIα alone or in complex with the DNA repair scaffold protein XRCC1. Small-angle X-ray scattering and negative stain electron microscopy combined with mapping of the interacting regions identified a TDP1/LigIIIα compact dimer of heterodimers in which the two LigIII catalytic cores are positioned in the center, whereas the two TDP1 molecules are located at the edges of the core complex flanked by highly flexible regions that can interact with other repair proteins and SSBs. As TDP1and LigIIIα together repair adducts caused by TOP1 cancer chemotherapy inhibitors, the defined interaction architecture and regulation of this enzyme complex provide insights into a key repair pathway in nonmalignant and cancer cells

Amphipol-Trapped ExbB–ExbD Membrane Protein Complex from Escherichia coli: A Biochemical and Structural Case Study

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An atypical BRCT-BRCT interaction with the XRCC1 scaffold protein compacts human DNA Ligase IIIα within a flexible DNA repair complex.

The XRCC1-DNA ligase IIIα complex (XL) is critical for DNA single-strand break repair, a key target for PARP inhibitors in cancer cells deficient in homologous recombination. Here, we combined biophysical approaches to gain insights into the shape and conformational flexibility of the XL as well as XRCC1 and DNA ligase IIIα (LigIIIα) alone. Structurally-guided mutational analyses based on the crystal structure of the human BRCT-BRCT heterodimer identified the network of salt bridges that together with the N-terminal extension of the XRCC1 C-terminal BRCT domain constitute the XL molecular interface. Coupling size exclusion chromatography with small angle X-ray scattering and multiangle light scattering (SEC-SAXS-MALS), we determined that the XL is more compact than either XRCC1 or LigIIIα, both of which form transient homodimers and are highly disordered. The reduced disorder and flexibility allowed us to build models of XL particles visualized by negative stain electron microscopy that predict close spatial organization between the LigIIIα catalytic core and both BRCT domains of XRCC1. Together our results identify an atypical BRCT-BRCT interaction as the stable nucleating core of the XL that links the flexible nick sensing and catalytic domains of LigIIIα to other protein partners of the flexible XRCC1 scaffold