European Molecular Biology Laboratory

The InterPro database (http://www.ebi.ac.uk/interpro/) is a freely available resource that can be used to classify sequences into protein families and to predict the presence of important domains and sites. Central to the InterPro database are predictive models, known as signatures, from a range of different protein family databases that have different biological focuses and use different methodological approaches to classify protein families and domains. InterPro integrates these signatures, capitalizing on the respective strengths of the individual databases, to produce a powerful protein classification resource. Here, we report on the status of InterPro as it enters its 15th year of operation, and give an overview of new developments with the database and its associated Web interfaces and software. In particular, the new domain architecture search tool is described and the process of mapping of Gene Ontology terms to InterPro is outlined. We also discuss the challenges faced by the resource given the explosive growth in sequence data in recent years. InterPro (version 48.0) contains 36,766 member database signatures integrated into 26,238 InterPro entries, an increase of over 3993 entries (5081 signatures), since 2012.

Structural analysis of flexible macromolecular systems such as intrinsically disordered or multidomain proteins with flexible linkers is a difficult task as high-resolution techniques are barely applicable. A new approach, ensemble optimization method (EOM), is proposed to quantitatively characterize flexible proteins in solution using small-angle X-ray scattering (SAXS). The flexibility is taken into account by allowing for the coexistence of different conformations of the protein contributing to the experimental scattering pattern. These conformers are selected using a genetic algorithm from a pool containing a large number of randomly generated models covering the protein configurational space. Quantitative criteria are developed to analyze the EOM selected models and to determine the optimum number of conformers in the ensemble. Simultaneous fitting of multiple scattering patterns from deletion mutants, if available, provides yet more detailed local information about the structure. The efficiency of EOM is demonstrated in model and practical examples on completely or partially unfolded proteins and on multidomain proteins interconnected by linkers. In the latter case, EOM is able to distinguish between rigid and flexible proteins and to directly assess the interdomain contacts.

The structure of the protein-solvent interface is the subject of controversy in theoretical studies and requires direct experimental characterization. Three proteins with known atomic resolution crystal structure (lysozyme, Escherichia coli thioredoxin reductase, and protein R1 of E. coli ribonucleotide reductase) were investigated in parallel by x-ray and neutron scattering in H2O and D2O solutions. The analysis of the protein-solvent interface is based on the significantly different contrasts for the protein and for the hydration shell. The results point to the existence of a first hydration shell with an average density approximately 10% larger than that of the bulk solvent in the conditions studied. Comparisons with the results of other studies suggest that this may be a general property of aqueous interfaces.

Aminoacyl-transfer RNA (tRNA) synthetases, which catalyze the attachment of the correct amino acid to its corresponding tRNA during translation of the genetic code, are proven antimicrobial drug targets. We show that the broad-spectrum antifungal 5-fluoro-1,3-dihydro-1-hydroxy-2,1-benzoxaborole (AN2690), in development for the treatment of onychomycosis, inhibits yeast cytoplasmic leucyl-tRNA synthetase by formation of a stable tRNA(Leu)-AN2690 adduct in the editing site of the enzyme. Adduct formation is mediated through the boron atom of AN2690 and the 2'- and 3'-oxygen atoms of tRNA's3'-terminal adenosine. The trapping of enzyme-bound tRNA(Leu) in the editing site prevents catalytic turnover, thus inhibiting synthesis of leucyl-tRNA(Leu) and consequentially blocking protein synthesis. This result establishes the editing site as a bona fide target for aminoacyl-tRNA synthetase inhibitors.

Abscisic acid (ABA) is a key phytohormone involved in adaption to environmental stress and regulation of plant development. Clade A protein phosphatases type 2C (PP2Cs), such as HAB1, are key negative regulators of ABA signaling in Arabidopsis. To obtain further insight into regulation of HAB1 function by ABA, we have screened for HAB1-interacting partners using a yeast two-hybrid approach. Three proteins were identified, PYL5, PYL6 and PYL8, which belong to a 14-member subfamily of the Bet v1-like superfamily. HAB1-PYL5 interaction was confirmed using BiFC and co-immunoprecipitation assays. PYL5 over-expression led to a globally enhanced response to ABA, in contrast to the opposite phenotype reported for HAB1-over-expressing plants. F(2) plants that over-expressed both HAB1 and PYL5 showed an enhanced response to ABA, indicating that PYL5 antagonizes HAB1 function. PYL5 and other members of its protein family inhibited HAB1, ABI1 and ABI2 phosphatase activity in an ABA-dependent manner. Isothermal titration calorimetry revealed saturable binding of (+)ABA to PYL5, with K(d) values of 1.1 mum or 38 nm in the absence or presence of the PP2C catalytic core of HAB1, respectively. Our work indicates that PYL5 is a cytosolic and nuclear ABA receptor that activates ABA signaling through direct inhibition of clade A PP2Cs. Moreover, we show that enhanced resistance to drought can be obtained through PYL5-mediated inhibition of clade A PP2Cs.

Type 1 pili—adhesive fibers expressed in most members of the Enterobacteriaceae family—mediate binding to mannose receptors on host cells through the FimH adhesin. Pilus biogenesis proceeds by way of the chaperone/usher pathway. The x-ray structure of the FimC-FimH chaperone-adhesin complex from uropathogenic Escherichia coli at 2.5 angstrom resolution reveals the basis for carbohydrate recognition and for pilus assembly. The carboxyl-terminal pilin domain of FimH has an immunoglobulin-like fold, except that the seventh strand is missing, leaving part of the hydrophobic core exposed. A donor strand complementation mechanism in which the chaperone donates a strand to complete the pilin domain explains the basis for both chaperone function and pilus biogenesis.

A high-brilliance synchrotron P12 beamline of the EMBL located at the PETRA III storage ring (DESY, Hamburg) is dedicated to biological small-angle X-ray scattering (SAXS) and has been designed and optimized for scattering experiments on macromolecular solutions. Scatterless slits reduce the parasitic scattering, a custom-designed miniature active beamstop ensures accurate data normalization and the photon-counting PILATUS 2M detector enables the background-free detection of weak scattering signals. The high flux and small beam size allow for rapid experiments with exposure time down to 30-50 ms covering the resolution range from about 300 to 0.5 nm. P12 possesses a versatile and flexible sample environment system that caters for the diverse experimental needs required to study macromolecular solutions. These include an in-vacuum capillary mode for standard batch sample analyses with robotic sample delivery and for continuous-flow in-line sample purification and characterization, as well as an in-air capillary time-resolved stopped-flow setup. A novel microfluidic centrifugal mixing device (SAXS disc) is developed for a high-throughput screening mode using sub-microlitre sample volumes. Automation is a key feature of P12; it is controlled by a beamline meta server, which coordinates and schedules experiments from either standard or nonstandard operational setups. The integrated SASFLOW pipeline automatically checks for consistency, and processes and analyses the data, providing near real-time assessments of overall parameters and the generation of low-resolution models within minutes of data collection. These advances, combined with a remote access option, allow for rapid high-throughput analysis, as well as time-resolved and screening experiments for novice and expert biological SAXS users.

The unusual adsorption behavior of CO2 in a nanoporous hybrid metal– organic solid is discussed (see figure). The results indicate that the gas adsorption–desorption step is related to a breathing phenomenon. This study also suggests that the main interactions responsible for the breathing phenomenon are strong guest–framework CO2–OH interactions as well as CO2–CO2 interactions along the tunnels present in the structure. Supporting information for this article is available on the WWW under http://www.wiley-vch.de/contents/jc_2089/2007/c2645_s.pdf or from the author. Please note: The publisher is not responsible for the content or functionality of any supporting information supplied by the authors. Any queries (other than missing content) should be directed to the corresponding author for the article.

Midzone microtubules of mammalian cells play an essential role in the induction of cell cleavage, serving as a platform for a number of proteins that play a part in cytokinesis. We demonstrate that PRC1, a mitotic spindle-associated Cdk substrate that is essential to cell cleavage, is a microtubule binding and bundling protein both in vivo and in vitro. Overexpression of PRC1 extensively bundles interphase microtubules, but does not affect early mitotic spindle organization. PRC1 contains two Cdk phosphorylation motifs, and phosphorylation is possibly important to mitotic suppression of bundling, as a Cdk phosphorylation-null mutant causes extensive bundling of the prometaphase spindle. Complete suppression of PRC1 by siRNA causes failure of microtubule interdigitation between half spindles and the absence of a spindle midzone. Truncation mutants demonstrate that the NH2-terminal region of PRC1, rich in alpha-helical sequence, is important for localization to the cleavage furrow and to the center of the midbody, whereas the central region, with the highest sequence homology between species, is required for microtubule binding and bundling activity. We conclude that PRC1 is a microtubule-associated protein required to maintain the spindle midzone, and that distinct functions are associated with modular elements of the primary sequence.

Synchrotron light source facilities worldwide generate terabytes of data in numerous incompatible data formats from a wide range of experiment types. The Data Analysis WorkbeNch (DAWN) was developed to address the challenge of providing a single visualization and analysis platform for data from any synchrotron experiment (including single-crystal and powder diffraction, tomography and spectroscopy), whilst also being sufficiently extensible for new specific use case analysis environments to be incorporated (e.g. ARPES, PEEM). In this work, the history and current state of DAWN are presented, with two case studies to demonstrate specific functionality. The first is an example of a data processing and reduction problem using the generic tools, whilst the second shows how these tools can be targeted to a specific scientific area.

The crystal structure of Thermus thermophilus seryl-transfer RNA synthetase, a class 2 aminoacyl-tRNA synthetase, complexed with a single tRNA(Ser) molecule was solved at 2.9 A resolution. The structure revealed how insertion of conserved base G20b from the D loop into the core of the tRNA determines the orientation of the long variable arm, which is a characteristic feature of most serine specific tRNAs. On tRNA binding, the antiparallel coiled-coil domain of one subunit of the synthetase makes contacts with the variable arm and T psi C loop of the tRNA and directs the acceptor stem of the tRNA into the active site of the other subunit. Specificity depends principally on recognition of the shape of tRNA(Ser) through backbone contacts and secondarily on sequence specific interactions.

Radiation damage is an inherent problem in x-ray crystallography. It usually is presumed to be nonspecific and manifested as a gradual decay in the overall quality of data obtained for a given crystal as data collection proceeds. Based on third-generation synchrotron x-ray data, collected at cryogenic temperatures, we show for the enzymes Torpedo californica acetylcholinesterase and hen egg white lysozyme that synchrotron radiation also can cause highly specific damage. Disulfide bridges break, and carboxyl groups of acidic residues lose their definition. Highly exposed carboxyls, and those in the active site of both enzymes, appear particularly susceptible. The catalytic triad residue, His-440, in acetylcholinesterase, also appears to be much more sensitive to radiation damage than other histidine residues. Our findings have direct practical implications for routine x-ray data collection at high-energy synchrotron sources. Furthermore, they provide a direct approach for studying the radiation chemistry of proteins and nucleic acids at a detailed, structural level and also may yield information concerning putative "weak links" in a given biological macromolecule, which may be of structural and functional significance.

Natively unfolded proteins play key roles in normal and pathological biochemical processes. Despite their importance for function, this category of proteins remains beyond the reach of classical structural biology because of their inherent conformational heterogeneity. We present a description of the intrinsic conformational sampling of unfolded proteins based on residue-specific /Psi propensities from loop regions of a folded protein database and simple volume exclusion. This approach is used to propose a structural model of the 57-aa, natively disordered region of the nucleocapsid-binding domain of Sendai virus phosphoprotein. Structural ensembles obeying these simple rules of conformational sampling are used to simulate averaged residual dipolar couplings (RDCs) and small-angle x-ray scattering data. This protein is particularly informative because RDC data from the equally sized folded and unfolded domains both report on the unstructured region, allowing a quantitative analysis of the degree of order present in this part of the protein. Close agreement between experimental and simulated RDC and small-angle x-ray scattering data validates this simple model of conformational sampling, providing a precise description of local structure and dynamics and average dimensions of the ensemble of sampled structures. RDC data from two urea-unfolded systems are also closely reproduced. The demonstration that conformational behavior of unfolded proteins can be accurately predicted from the primary sequence by using a simple set of rules has important consequences for our understanding of the structure and dynamics of the unstructured state.

Abstract Baloxavir acid (BXA), derived from the prodrug baloxavir marboxil (BXM), potently and selectively inhibits the cap-dependent endonuclease within the polymerase PA subunit of influenza A and B viruses. In clinical trials, single doses of BXM profoundly decrease viral titers as well as alleviating influenza symptoms. Here, we characterize the impact on BXA susceptibility and replicative capacity of variant viruses detected in the post-treatment monitoring of the clinical studies. We find that the PA I38T substitution is a major pathway for reduced susceptibility to BXA, with 30- to 50-fold and 7-fold EC 50 changes in A and B viruses, respectively. The viruses harboring the I38T substitution show severely impaired replicative fitness in cells, and correspondingly reduced endonuclease activity in vitro . Co-crystal structures of wild-type and I38T influenza A and B endonucleases bound to BXA show that the mutation reduces van der Waals contacts with the inhibitor. A reduced affinity to the I38T mutant is supported by the lower stability of the BXA-bound endonuclease. These mechanistic insights provide markers for future surveillance of treated populations.

Small-angle X-ray scattering (SAXS) measurements of proteins in solution are becoming increasingly popular with biochemists and structural biologists owing to the presence of dedicated high-throughput beamlines at synchrotron sources. As part of the ESRF Upgrade program a dedicated instrument for performing SAXS from biological macromolecules in solution (BioSAXS) has been installed at the renovated BM29 location. The optics hutch has been equipped with new optical components of which the two principal elements are a fixed-exit double multilayer monochromator and a 1.1 m-long toroidal mirror. These new dedicated optics give improved beam characteristics (compared with the previous set-up on ID14-3) regarding the energy tunability, flux and focusing at the detector plane leading to reduced parasitic scattering and an extended s-range. User experiments on the beamline have been successfully carried out since June 2012. A description of the new BioSAXS beamline and the set-up characteristics are presented together with examples of obtained data.

A RNA methyltransferase in facilitating early development via regulation of SAM availability.

Negative-strand RNA viruses condense their genome into a helical nucleoprotein-RNA complex, the nucleocapsid, which is packed into virions and serves as a template for the RNA-dependent RNA polymerase complex. The crystal structure of a recombinant rabies virus nucleoprotein-RNA complex, organized in an undecameric ring, has been determined at 3.5 angstrom resolution. Polymerization of the nucleoprotein is achieved by domain exchange between protomers, with flexible hinges allowing nucleocapsid formation. The two core domains of the nucleoprotein clamp around the RNA at their interface and shield it from the environment. RNA sequestering by nucleoproteins is likely a common mechanism used by negative-strand RNA viruses to protect their genomes from the innate immune response directed against viral RNA in human host cells at certain stages of an infectious cycle.

The mechanisms by which intrinsically disordered proteins engage in rapid and highly selective binding is a subject of considerable interest and represents a central paradigm to nuclear pore complex (NPC) function, where nuclear transport receptors (NTRs) move through the NPC by binding disordered phenylalanine-glycine-rich nucleoporins (FG-Nups). Combining single-molecule fluorescence, molecular simulations, and nuclear magnetic resonance, we show that a rapidly fluctuating FG-Nup populates an ensemble of conformations that are prone to bind NTRs with near diffusion-limited on rates, as shown by stopped-flow kinetic measurements. This is achieved using multiple, minimalistic, low-affinity binding motifs that are in rapid exchange when engaging with the NTR, allowing the FG-Nup to maintain an unexpectedly high plasticity in its bound state. We propose that these exceptional physical characteristics enable a rapid and specific transport mechanism in the physiological context, a notion supported by single molecule in-cell assays on intact NPCs.

In plants, the AUXIN RESPONSE FACTOR (ARF) transcription factor family regulates gene expression in response to auxin. In the absence of auxin, ARF transcription factors are repressed by interaction with AUXIN/INDOLE 3-ACETIC ACID (Aux/IAA) proteins. Although the C termini of ARF and Aux/IAA proteins facilitate their homo- and heterooligomerization, the molecular basis for this interaction remained undefined. The crystal structure of the C-terminal interaction domain of Arabidopsis ARF7 reveals a Phox and Bem1p (PB1) domain that provides both positive and negative electrostatic interfaces for directional protein interaction. Mutation of interface residues in the ARF7 PB1 domain yields monomeric protein and abolishes interaction with both itself and IAA17. Expression of a stabilized Aux/IAA protein (i.e., IAA16) bearing PB1 mutations in Arabidopsis suggests a multimerization requirement for ARF protein repression, leading to a refined auxin-signaling model.

Toxoplasma gondii, the causative agent of toxoplasmosis, is an obligate intracellular protozoan parasite that resides inside a parasitophorous vacuole. During infection, Toxoplasma actively remodels the transcriptome of its hosting cells with profound and coupled impact on the host immune response. We report that Toxoplasma secretes GRA24, a novel dense granule protein which traffics from the vacuole to the host cell nucleus. Once released into the host cell, GRA24 has the unique ability to trigger prolonged autophosphorylation and nuclear translocation of the host cell p38α MAP kinase. This noncanonical kinetics of p38α activation correlates with the up-regulation of the transcription factors Egr-1 and c-Fos and the correlated synthesis of key proinflammatory cytokines, including interleukin-12 and the chemokine MCP-1, both known to control early parasite replication in vivo. Remarkably, the GRA24-p38α complex is defined by peculiar structural features and uncovers a new regulatory signaling path distinct from the MAPK signaling cascade and otherwise commonly activated by stress-related stimuli or various intracellular microbes.