Efficient determination of the accessible conformation space of multi-domain complexes based on EPR PELDOR data

Many proteins can adopt multiple conformations which are important for their function. This is also true for proteins and domains that are covalently linked to each other. One important example is ubiquitin, which can form chains of different conformations depending on which of its lysine side chains is used to form an isopeptide bond with the C-terminus of another ubiquitin molecule. Similarly, ubiquitin gets covalently attached to active-site residues of E2 ubiquitin-conjugating enzymes. Due to weak interactions between ubiquitin and its interaction partners, these covalent complexes adopt multiple conformations. Understanding the function of these complexes requires the characterization of the entire accessible conformation space and its modulation by interaction partners. Long-range (1.8-10 nm) distance restraints obtained by EPR spectroscopy in the form of probability distributions are ideally suited for this task as not only the mean distance but also information about the conformation dynamics is encoded in the experimental data. Here we describe a computational method that we have developed based on well-established structure determination software using NMR restraints to calculate the accessible conformation space using PELDOR/DEER data

The UBA domain of conjugating enzyme Ubc1/Ube2K facilitates assembly of K48/K63‐branched ubiquitin chains

The assembly of a specific polymeric ubiquitin chain on a target protein is a key event in the regulation of numerous cellular processes. Yet, the mechanisms that govern the selective synthesis of particular polyubiquitin signals remain enigmatic. The homologous ubiquitin‐conjugating (E2) enzymes Ubc1 (budding yeast) and Ube2K (mammals) exclusively generate polyubiquitin linked through lysine 48 (K48). Uniquely among E2 enzymes, Ubc1 and Ube2K harbor a ubiquitin‐binding UBA domain with unknown function. We found that this UBA domain preferentially interacts with ubiquitin chains linked through lysine 63 (K63). Based on structural modeling, in vitro ubiquitination experiments, and NMR studies, we propose that the UBA domain aligns Ubc1 with K63‐linked polyubiquitin and facilitates the selective assembly of K48/K63‐branched ubiquitin conjugates. Genetic and proteomics experiments link the activity of the UBA domain, and hence the formation of this unusual ubiquitin chain topology, to the maintenance of cellular proteostasis

K48- and K63-linked ubiquitin chain interactome reveals branch- and length-specific ubiquitin interactors

The ubiquitin (Ub) code denotes the complex Ub architectures, including Ub chains of different lengths, linkage types, and linkage combinations, which enable ubiquitination to control a wide range of protein fates. Although many linkage-specific interactors have been described, how interactors are able to decode more complex architectures is not fully understood. We conducted a Ub interactor screen, in humans and yeast, using Ub chains of varying lengths, as well as homotypic and heterotypic branched chains of the two most abundant linkage types—lysine 48–linked (K48) and lysine 63–linked (K63) Ub. We identified some of the first K48/K63-linked branch-specific Ub interactors, including histone ADP-ribosyltransferase PARP10/ARTD10, E3 ligase UBR4, and huntingtin-interacting protein HIP1. Furthermore, we revealed the importance of chain length by identifying interactors with a preference for Ub3 over Ub2 chains, including Ub-directed endoprotease DDI2, autophagy receptor CCDC50, and p97 adaptor FAF1. Crucially, we compared datasets collected using two common deubiquitinase inhibitors—chloroacetamide and N-ethylmaleimide. This revealed inhibitor-dependent interactors, highlighting the importance of inhibitor consideration during pulldown studies. This dataset is a key resource for understanding how the Ub code is read

Test von C/SiC-Faserkeramik-Hitzeschutzmaterialproben waehrend eines realen Wiedereintritts auf FOTON Endbericht

The DLR Stuttgart manufactured two flat disk-like samples of non-ablative heat protection material. These were integrated into the ablative heat protection system of FOTON-8 by KB FOTON. After the mission and re-entry of the capsule the samples, which were positioned near the stagnation point of the capsule and thus exposed to the maximum heat load, were deintegrated and analyzed afterwards. There could be seen no damages or essential changes of the samples. Thus the German material was successfully exposed to a real re-entry for the first time. We want to point out that the material remained absolutely stable at the contact area to the ablative Russian material. This proves the compatibility between the different materials and demonstrates that the integration of the ceramic tile to the nose of the EXPRESS capsule should be possible and not too risky as it is actually planned. (orig.)Es wurden zwei scheibenfoermige Probenstuecke aus nicht-ablativem Hitzeschutzmaterial aus C/SiC-Faserkeramik hergestellt und von KB FOTON in den ablativen Hitzeschutz der FOTON-8-Kapsel integriert. Nach dem Flug und Wiedereintritt der Kapsel wurden die Proben, die nahe des Staupunktes der maximalen Last ausgesetzt waren, deintegriert und anschliessend analysiert. Es konnten keine wesentlichen Schaedigungen oder Veraenderungen festgestellt werden. Damit wurde erstmalig dieses Material erfolgreich einem realen Wiedereintritt ausgesetzt, wobei hervorzuheben ist, dass das Material auch an der Kontaktstelle zum ablativen, russischen Hitzeschutzmaterial voellig stabil geblieben ist. Dies zeigt, dass die Kompatibilitaet zwischen dem russischen und dem deutschen Material gegeben ist, und dass die Integration der Faserkeramikkachel im Staupunkt der EXPRESS-Kapsel wie geplant moeglich sein und kein zu hohes Risiko in sich bergen sollte. (orig.)SIGLEAvailable from TIB Hannover: F95B557+a / FIZ - Fachinformationszzentrum Karlsruhe / TIB - Technische InformationsbibliothekBundesministerium fuer Forschung und Technologie (BMFT), Bonn (Germany); Deutsche Agentur fuer Raumfahrtangelegenheiten (DARA) GmbH, Bonn (Germany)DEGerman

Chain assembly and disassembly processes differently affect the conformational space of ubiquitin chains

Ubiquitination is the most versatile posttranslational modification. The information is encoded by linkage type as well as chain length, which are translated by ubiquitin binding domains into specific signaling events. Chain topology determines the conformational space of a ubiquitin chain and adds an additional regulatory layer to this ubiquitin code. In particular, processes that modify chain length will be affected by chain conformations as they require access to the elongation or cleavage sites. We investigated conformational distributions in the context of chain elongation and disassembly using pulsed electron-electron double resonance spectroscopy in combination with molecular modeling. Analysis of the conformational space of diubiquitin revealed conformational selection or remodeling as mechanisms for chain recognition during elongation or hydrolysis, respectively. Chain elongation to tetraubiquitin increases the sampled conformational space, suggesting that a high intrinsic flexibility of K48-linked chains may contribute to efficient proteasomal degradation