Molecular Mechanism of J-Domain-Triggered ATP Hydrolysis by Hsp70 Chaperones.

Efficient targeting of Hsp70 chaperones to substrate proteins depends on J-domain cochaperones, which in synergism with substrates trigger ATP hydrolysis in Hsp70s and concomitant substrate trapping. We present the crystal structure of the J-domain of Escherichia coli DnaJ in complex with the E. coli Hsp70 DnaK. The J-domain interacts not only with DnaK's nucleotide-binding domain (NBD) but also with its substrate-binding domain (SBD) and packs against the highly conserved interdomain linker. Mutational replacement of contacts between J-domain and SBD strongly reduces the ability of substrates to stimulate ATP hydrolysis in the presence of DnaJ and compromises viability at heat shock temperatures. Our data demonstrate that the J-domain and the substrate do not deliver completely independent signals for ATP hydrolysis, but the J-domain, in addition to its direct influence on Hsp70s catalytic center, makes Hsp70 more responsive for the hydrolysis-inducing signal of the substrate, resulting in efficient substrate trapping.

Extended N-Terminal Acetyltransferase Naa50 in Filamentous Fungi Adds to Naa50 Diversity.

Most eukaryotic proteins are N-terminally acetylated by a set of Nα acetyltransferases (NATs). This ancient and ubiquitous modification plays a fundamental role in protein homeostasis, while mutations are linked to human diseases and phenotypic defects. In particular, Naa50 features species-specific differences, as it is inactive in yeast but active in higher eukaryotes. Together with NatA, it engages in NatE complex formation for cotranslational acetylation. Here, we report Naa50 homologs from the filamentous fungi Chaetomium thermophilum and Neurospora crassa with significant N- and C-terminal extensions to the conserved GNAT domain. Structural and biochemical analyses show that CtNaa50 shares the GNAT structure and substrate specificity with other homologs. However, in contrast to previously analyzed Naa50 proteins, it does not form NatE. The elongated N-terminus increases Naa50 thermostability and binds to dynein light chain protein 1, while our data suggest that conserved positive patches in the C-terminus allow for ribosome binding independent of NatA. Our study provides new insights into the many facets of Naa50 and highlights the diversification of NATs during evolution.

Development of operational intervention levels for events with release of radionuclides.

An essential part of a protection strategy for radiological emergencies is the development of national dose criteria and of operational intervention levels (OILs) to decide about protective measures for all ten scenarios Germany is preparing. For the process of planning and implementing such protection strategies as required by the German Radiation Protection Law the Federal Ministry BMU has commissioned the German Radiation Protection Commission (SSK) to recommend dose criteria and OILs for emergency response measures. OILs link a chosen dose criterion for a protective action with a suitable measurement of the contamination situation such as ambient dose rate (µSv h-1), contamination level on surfaces (Bq cm-2) or activity content (Bq g-1, Bq cm-3). This link should adequately model the exposure of persons during a defined exposure period (e.g. seven days, one year) caused by the measured contamination. Dose calculations to quantify OILs should apply assumptions and parameter values that are in tendency realistic and not unduly conservative. OILs have been developed for the following emergency response actions based on radiation measurements:Sheltering on the basis of dose rate (µSv h-1) and contamination level(Bq cm-2).Evacuation on the basis of dose rate (µSv h-1) and contamination level(Bq cm-2).Establishing a radiological hazard area to implement access andcontamination control on the basis of dose rate (µSv h-1) and contaminationlevel (Bq cm-2).Contamination control and possibly decontamination of persons and objects(items, goods, vehicles, etc) based on contamination level (Bq cm-2).A set of precautionary early actions: warning the population not toconsume freshly contaminated food and agricultural measures to reducefood contamination based on dose rate (µSv h-1).Application of maximum permitted levels of radioactive contamination offood and feed (Bq kg-1) according to Euratom Regulation.

The Arabidopsis Nα -acetyltransferase NAA60 locates to the plasma membrane and is vital for the high salt stress response.

In humans and plants, N-terminal acetylation plays a central role in protein homeostasis, affects 80% of proteins in the cytoplasm and is catalyzed by five ribosome-associated N-acetyltransferases (NatA-E). Humans also possess a Golgi-associated NatF (HsNAA60) that is essential for Golgi integrity. Remarkably, NAA60 is absent in fungi and has not been identified in plants. Here we identify and characterize the first plasma membrane-anchored post-translationally acting N-acetyltransferase AtNAA60 in the reference plant Arabidopsis thaliana by the combined application of reverse genetics, global proteomics, live-cell imaging, microscale thermophoresis, circular dichroism spectroscopy, nano-differential scanning fluorometry, intrinsic tryptophan fluorescence and X-ray crystallography. We demonstrate that AtNAA60, like HsNAA60, is membrane-localized in vivo by an α-helical membrane anchor at its C-terminus, but in contrast to HsNAA60, AtNAA60 localizes to the plasma membrane. The AtNAA60 crystal structure provides insights into substrate-binding, the broad substrate specificity and the catalytic mechanism probed by structure-based mutagenesis. Characterization of the NAA60 loss-of-function mutants (naa60-1 and naa60-2) uncovers a plasma membrane-localized substrate of AtNAA60 and the importance of NAA60 during high salt stress. Our findings provide evidence for the plant-specific evolution of a plasma membrane-anchored N-acetyltransferase that is vital for adaptation to stress.

Structure and dynamics of the ATP-bound open conformation of Hsp70 chaperones.

Central to the chaperone function of Hsp70s is the transition between open and closed conformations of their polypeptide substrate binding domain (SBD), which is regulated through an allosteric mechanism via ATP binding and hydrolysis in their nucleotide binding domain (NBD). Although the structure of the closed conformation of Hsp70s is well studied, the open conformation has remained elusive. Here, we report on the 2.4 Å crystal structure of the ATP-bound open conformation of the Escherichia coli Hsp70 homolog DnaK. In the open DnaK structure, the ß sheet and α-helical lid subdomains of the SBD are detached from one another and docked to different faces of the NBD. The contacts between the ß sheet subdomain and the NBD reveal the mechanism of allosteric regulation. In addition, we demonstrate that docking of the ß sheet and α-helical lid subdomains to the NBD is a sequential process influenced by peptide and protein substrates.

Structural and mechanistic insights into activation of the human RNA ligase RTCB by Archease.

RNA ligases of the RTCB-type play an essential role in tRNA splicing, the unfolded protein response and RNA repair. RTCB is the catalytic subunit of the pentameric human tRNA ligase complex. RNA ligation by the tRNA ligase complex requires GTP-dependent activation of RTCB. This active site guanylylation reaction relies on the activation factor Archease. The mechanistic interplay between both proteins has remained unknown. Here, we report a biochemical and structural analysis of the human RTCB-Archease complex in the pre- and post-activation state. Archease reaches into the active site of RTCB and promotes the formation of a covalent RTCB-GMP intermediate through coordination of GTP and metal ions. During the activation reaction, Archease prevents futile RNA substrate binding to RTCB. Moreover, monomer structures of Archease and RTCB reveal additional states within the RNA ligation mechanism. Taken together, we present structural snapshots along the reaction cycle of the human tRNA ligase.

Molecular basis for the unique role of the AAA+ chaperone ClpV in type VI protein secretion.

Ring-forming AAA(+) ATPases act in a plethora of cellular processes by remodeling macromolecules. The specificity of individual AAA(+) proteins is achieved by direct or adaptor-mediated association with substrates via distinct recognition domains. We investigated the molecular basis of substrate interaction for Vibrio cholerae ClpV, which disassembles tubular VipA/VipB complexes, an essential step of type VI protein secretion and bacterial virulence. We identified the ClpV recognition site within VipB, showed that productive ClpV-VipB interaction requires the oligomeric state of both proteins, solved the crystal structure of a ClpV N-domain-VipB peptide complex, and verified the interaction surface by mutant analysis. Our results show that the substrate is bound to a hydrophobic groove, which is formed by the addition of a single α-helix to the core N-domain. This helix is absent from homologous N-domains, explaining the unique substrate specificity of ClpV. A limited interaction surface between both proteins accounts for the dramatic increase in binding affinity upon ATP-driven ClpV hexamerization and VipA/VipB tubule assembly by coupling multiple weak interactions. This principle ensures ClpV selectivity toward the VipA/VipB macromolecular complex.

The protein structure initiative structural genomics knowledgebase.

The Protein Structure Initiative Structural Genomics Knowledgebase (PSI SGKB, http://kb.psi-structuralgenomics.org) has been created to turn the products of the PSI structural genomics effort into knowledge that can be used by the biological research community to understand living systems and disease. This resource provides central access to structures in the Protein Data Bank (PDB), along with functional annotations, associated homology models, worldwide protein target tracking information, available protocols and the potential to obtain DNA materials for many of the targets. It also offers the ability to search all of the structural and methodological publications and the innovative technologies that were catalyzed by the PSI's high-throughput research efforts. In collaboration with the Nature Publishing Group, the PSI SGKB provides a research library, editorials about new research advances, news and an events calendar to present a broader view of structural biology and structural genomics. By making these resources freely available, the PSI SGKB serves as a bridge to connect the structural biology and the greater biomedical communities.

Structural basis of Naa20 activity towards a canonical NatB substrate.

N-terminal acetylation is one of the most common protein modifications in eukaryotes and is carried out by N-terminal acetyltransferases (NATs). It plays important roles in protein homeostasis, localization, and interactions and is linked to various human diseases. NatB, one of the major co-translationally active NATs, is composed of the catalytic subunit Naa20 and the auxiliary subunit Naa25, and acetylates about 20% of the proteome. Here we show that NatB substrate specificity and catalytic mechanism are conserved among eukaryotes, and that Naa20 alone is able to acetylate NatB substrates in vitro. We show that Naa25 increases the Naa20 substrate affinity, and identify residues important for peptide binding and acetylation activity. We present the first Naa20 crystal structure in complex with the competitive inhibitor CoA-Ac-MDEL. Our findings demonstrate how Naa20 binds its substrates in the absence of Naa25 and support prospective endeavors to derive specific NAT inhibitors for drug development.

Structural and functional characterization of the N-terminal acetyltransferase Naa50.

The majority of eukaryotic proteins is modified by N-terminal acetylation, which plays a fundamental role in protein homeostasis, localization, and complex formation. N-terminal acetyltransferases (NATs) mainly act co-translationally on newly synthesized proteins at the ribosomal tunnel exit. NatA is the major NAT consisting of Naa10 catalytic and Naa15 auxiliary subunits, and with Naa50 forms the NatE complex. Naa50 has recently been identified in Arabidopsis thaliana and is important for plant development and stress response regulation. Here, we determined high-resolution X-ray crystal structures of AtNaa50 in complex with AcCoA and a bisubstrate analog. We characterized its substrate specificity, determined its enzymatic parameters, and identified functionally important residues. Even though Naa50 is conserved among species, we highlight differences between Arabidopsis and yeast, where Naa50 is catalytically inactive but binds CoA conjugates. Our study provides insights into Naa50 conservation, species-specific adaptations, and serves as a basis for further studies of NATs in plants.

The ribosome-associated complex RAC serves in a relay that directs nascent chains to Ssb.

The conserved ribosome-associated complex (RAC) consisting of Zuo1 (Hsp40) and Ssz1 (non-canonical Hsp70) acts together with the ribosome-bound Hsp70 chaperone Ssb in de novo protein folding at the ribosomal tunnel exit. Current models suggest that the function of Ssz1 is confined to the support of Zuo1, however, it is not known whether RAC by itself serves as a chaperone for nascent chains. Here we show that, via its rudimentary substrate binding domain (SBD), Ssz1 directly binds to emerging nascent chains prior to Ssb. Structural and biochemical analyses identify a conserved LP-motif at the Zuo1 N-terminus forming a polyproline-II helix, which binds to the Ssz1-SBD as a pseudo-substrate. The LP-motif competes with nascent chain binding to the Ssz1-SBD and modulates nascent chain transfer. The combined data indicate that Ssz1 is an active chaperone optimized for transient, low-affinity substrate binding, which ensures the flux of nascent chains through RAC/Ssb.

The Protein Model Portal.

Structural Genomics has been successful in determining the structures of many unique proteins in a high throughput manner. Still, the number of known protein sequences is much larger than the number of experimentally solved protein structures. Homology (or comparative) modeling methods make use of experimental protein structures to build models for evolutionary related proteins. Thereby, experimental structure determination efforts and homology modeling complement each other in the exploration of the protein structure space. One of the challenges in using model information effectively has been to access all models available for a specific protein in heterogeneous formats at different sites using various incompatible accession code systems. Often, structure models for hundreds of proteins can be derived from a given experimentally determined structure, using a variety of established methods. This has been done by all of the PSI centers, and by various independent modeling groups. The goal of the Protein Model Portal (PMP) is to provide a single portal which gives access to the various models that can be leveraged from PSI targets and other experimental protein structures. A single interface allows all existing pre-computed models across these various sites to be queried simultaneously, and provides links to interactive services for template selection, target-template alignment, model building, and quality assessment. The current release of the portal consists of 7.6 million model structures provided by different partner resources (CSMP, JCSG, MCSG, NESG, NYSGXRC, JCMM, ModBase, SWISS-MODEL Repository). The PMP is available at http://www.proteinmodelportal.org and from the PSI Structural Genomics Knowledgebase.

[Radiation exposure of staff during endovascular brachytherapy with Re-188 after PTA in the peripheral blood stream]. / Strahlenexposition des Personals bei der endovaskulären Brachytherapie (EVBT) mit Re-188 nach PTA im peripheren Stromgebiet.

Endovascular brachytherapy using a balloon catheter filled with Re-188 solution is a promising method for the prophylaxis of restenosis in peripheral blood circulation after percutaneous transluminal angioplasty (PTA) treatments. Thereby about 20 GBq Re-188 with a specific activity of about 5 GBq/ml are used. The high ionisation density of the beta radiation with high energy leads to selective irradiation of the blood vessel wall near the catheter, whereas the surrounding tissue remains almost unaffected. However the hospital staff has to carry out some work steps within close range to the high activity during preparation and therapy, causing a high risk of skin exposure, in particular at the hands. Estimations and measurements of the maximal local skin dose were made with thin-layered thermoluminescence dosimeters. It was assessed that the annual dose limit for skin of 500 mSv may be exceeded considerably when using conventional procedures and considering the expected number of 75 treatments per annum. By using the newly developed rhenium-188 application device "FlowMedical Application System" the exposure risk for the staff could be reduced drastically. The maximum skin dose of 76 mSv for the radiologist and of 50 mSv for the physicist was decreased to 2 mSv per treatment for both of them. Consequently, from the radiation protection point of view, the itm Rhenium-PTA is a safe method. Any exceeding of the dose limit can be prevented.

The SWISS-MODEL Repository: new features and functionalities.

The SWISS-MODEL Repository is a database of annotated 3D protein structure models generated by the SWISS-MODEL homology-modelling pipeline. As of September 2005, the repository contained 675,000 models for 604,000 different protein sequences of the UniProt database. Regular updates ensure that the content of the repository reflects the current state of sequence and structure databases, integrating new or modified target sequences, and making use of new template structures. Each Repository entry consists of one or more 3D models accompanied by detailed information about the target protein and the model building process: functional annotation, a detailed template selection log, target-template alignment, summary of the model building and model quality assessment. The SWISS-MODEL Repository is freely accessible at http://swissmodel.expasy.org/repository/.

Vitamin D deficiency may stimulate fibroblasts in Dupuytren's disease via mitochondrial increased reactive oxygen species through upregulating transforming growth factor-ß1.

Dupuytren's disease, a benign fibroproliferative disorder of the palmar fascia, represents an ideal model to study tissue fibrosis. Transforming growth factor-ß1 (TGF-ß1) and its downstream Smad signaling system is well established as a keyplayer during fibrogenesis. Vitamin D has been extensively studied as an anti-fibrotic agent in malignant chronic diseases. A number of studies have shown that myofibroblasts are main target cells of 1,25(OH)2D3 inhibitory action. The myofibroblast in the palmar aponeurosis of patients in different stages of Dupuytren's disease was found by electron microscopy to contain a large number of mitochondria. Mitochondria play a critical role in cell metabolism being the major source of reactive oxygen species (ROS) in cells. TGF-ß1 has been shown to increase mitochondrial ROS production in different cell types, which mediate fibrosis related gene expression and myofibroblast differentiation. TGF-ß1 increases mitochondrial ROS production in patients with Dupuytren's contracture potentially in consequence of Vitamin D deficiency, leading to myofibroblast differentiation. Thus, targeting this basic pathomechanism seems suitable to establish new treatment strategies.

Assessment of CASP7 predictions for template-based modeling targets.

This manuscript presents the assessment of the template-based modeling category of the seventh Critical Assessment of Techniques for Protein Structure Prediction (CASP7). The accuracy of predicted protein models for 108 target domains was assessed based on a detailed comparison between the experimental and predicted structures. The assessment was performed using numerical measures for backbone and structural alignment accuracy, and by scoring correctly modeled hydrogen bond interactions in the predictions. Based on these criteria, our statistical analysis identified a number of groups whose predictions were on average significantly more accurate. Furthermore, the predictions for six target proteins were evaluated for the accuracy of their modeled cofactor binding sites. We also assessed the ability of predictors to improve over the best available single template structure, which showed that the best groups produced models closer to the target structure than the best single template for a significant number of targets. In addition, we assessed the accuracy of the error estimates (local confidence values) assigned to predictions on a per residue basis. Finally, we discuss some general conclusions about the state of the art of template-based modeling methods and their usefulness for practical applications.

Domain definition and target classification for CASP7.

Experimentally determined protein structures formed the basis of the CASP7 prediction assessments. These target structures were assigned to one or more tertiary structure prediction categories and where necessary were divided into structural domains. Boundaries for these domains were based on visual inspection of the targets and superpositions of the target with template structures. Target domains were classified into three different categories for assessment: "high accuracy modeling," "template-based modeling," and "free modeling." Assessment categories were determined by structural similarity between the target domain and the nearest structural templates in the PDB and by the accuracy of the models submitted by the predictors or by whether or not template information was used to generate the predictions. In CASP7 108 of the 123 target domains were evaluated in the template-based modeling category and the remaining 15 target domains were classified as free modeling. A total of 28 target domains from the template-based modeling category were also assessed in the high accuracy category and four overlapped with the free modeling category.

Automated server predictions in CASP7.

With each round of CASP (Critical Assessment of Techniques for Protein Structure Prediction), automated prediction servers have played an increasingly important role. Today, most protein structure prediction approaches in some way depend on automated methods for fold recognition or model building. The accuracy of server predictions has significantly increased over the last years, and, in CASP7, we observed a continuation of this trend. In the template-based modeling category, the best prediction server was ranked third overall, i.e. it outperformed all but two of the human participating groups. This server also ranked among the very best predictors in the free modeling category as well, being clearly beaten by only one human group. In the high accuracy (HA) subset of TBM, two of the top five groups were servers. This article summarizes the contribution of automated structure prediction servers in the CASP7 experiment, with emphasis on 3D structure prediction, as well as information on their prediction scope and public availability.

Structural basis of HypK regulating N-terminal acetylation by the NatA complex.

In eukaryotes, N-terminal acetylation is one of the most common protein modifications involved in a wide range of biological processes. Most N-acetyltransferase complexes (NATs) act co-translationally, with the heterodimeric NatA complex modifying the majority of substrate proteins. Here we show that the Huntingtin yeast two-hybrid protein K (HypK) binds tightly to the NatA complex comprising the auxiliary subunit Naa15 and the catalytic subunit Naa10. The crystal structures of NatA bound to HypK or to a N-terminal deletion variant of HypK were determined without or with a bi-substrate analogue, respectively. The HypK C-terminal region is responsible for high-affinity interaction with the C-terminal part of Naa15. In combination with acetylation assays, the HypK N-terminal region is identified as a negative regulator of the NatA acetylation activity. Our study provides mechanistic insights into the regulation of this pivotal protein modification.