Conformational and functional characterization of artificially conjugated non-canonical ubiquitin dimers
2019-12-27, Schneider, Tobias, Berg, Andrej, Ulusoy, Zeynel, Gamerdinger, Martin, Peter, Christine, Kovermann, Michael
Ubiquitylation is an eminent posttranslational modification referring to the covalent attachment of single ubiquitin molecules or polyubiquitin chains to a target protein dictating the fate of such labeled polypeptide chains. Here, we have biochemically produced artificially Lys11-, and Lys27-, and Lys63-linked ubiquitin dimers based on click-chemistry generating milligram quantities in high purity. We show that the artificial linkage used for the conjugation of two ubiquitin moieties represents a fully reliable surrogate of the natural isopeptide bond by acquiring highly resolved nuclear magnetic resonance (NMR) spectroscopic data including ligand binding studies. Extensive coarse grained and atomistic molecular dynamics (MD) simulations allow to extract structures representing the ensemble of domain-domain conformations used to verify the experimental data. Advantageously, this methodology does not require individual isotopic labeling of both ubiquitin moieties as NMR data have been acquired on the isotopically labeled proximal moiety and complementary MD simulations have been used to fully interpret the experimental data in terms of domain-domain conformation. This combined approach intertwining NMR spectroscopy with MD simulations makes it possible to describe the conformational space non-canonically Lys11-, and Lys27-linked ubiquitin dimers occupy in a solution averaged ensemble by taking atomically resolved information representing all residues in ubiquitin dimers into account.
The structure of the ubiquitin-like modifier FAT10 reveals an alternative targeting mechanism for proteasomal degradation
2018-08-20, Aichem, Annette, Catone, Nicola, Berg, Andrej, Schwab, Ricarda, Scheuermann, Sophia, Bialas, Johanna, Schmidtke, Gunter, Peter, Christine, Gröttrup, Marcus, Wiesner, Silke
FAT10 is a ubiquitin-like modifier that directly targets proteins for proteasomal degradation. Here, we report the high-resolution structures of the two individual ubiquitin-like domains (UBD) of FAT10 that are joined by a flexible linker. While the UBDs of FAT10 show the typical ubiquitin-fold, their surfaces are entirely different from each other and from ubiquitin explaining their unique binding specificities. Deletion of the linker abrogates FAT10-conjugation while its mutation blocks auto-FAT10ylation of the FAT10-conjugating enzyme USE1 but not bulk conjugate formation. FAT10- but not ubiquitin-mediated degradation is independent of the segregase VCP/p97 in the presence but not the absence of FAT10's unstructured N-terminal heptapeptide. Stabilization of the FAT10 UBDs strongly decelerates degradation suggesting that the intrinsic instability of FAT10 together with its disordered N-terminus enables the rapid, joint degradation of FAT10 and its substrates without the need for FAT10 de-conjugation and partial substrate unfolding.
Simulating and analysing configurational landscapes of protein-protein contact formation
2019-06-06, Berg, Andrej, Peter, Christine
Interacting proteins can form aggregates and protein-protein interfaces with multiple patterns and different stabilities. Using molecular simulation one would like to understand the formation of these aggregates and which of the observed states are relevant for protein function and recognition. To characterize the complex configurational ensemble of protein aggregates, one needs a quantitative measure for the similarity of structures. We present well-suited descriptors that capture the essential features of non-covalent protein contact formation and domain motion. This set of collective variables is used with a nonlinear multi-dimensional scaling-based dimensionality reduction technique to obtain a low-dimensional representation of the configurational landscape of two ubiquitin proteins from coarse-grained simulations. We show that this two-dimensional representation is a powerful basis to identify meaningful states in the ensemble of aggregated structures and to calculate distributions and free energy landscapes for different sets of simulations. By using a measure to quantitatively compare free energy landscapes we can show how the introduction of a covalent bond between two ubiquitin proteins at different positions alters the configurational states of these dimers.
Towards a molecular basis of ubiquitin signaling : a dual-scale simulation study of ubiquitin dimers
2018-11, Berg, Andrej, Kukharenko, Oleksandra, Scheffner, Martin, Peter, Christine
Covalent modification of proteins by ubiquitin or ubiquitin chains is one of the most prevalent post-translational modifications in eukaryotes. Different types of ubiquitin chains are assumed to selectively signal respectively modified proteins for different fates. In support of this hypothesis, structural studies have shown that the eight possible ubiquitin dimers adopt different conformations. However, at least in some cases, these structures cannot sufficiently explain the molecular basis of the selective signaling mechanisms. This indicates that the available structures represent only a few distinct conformations within the entire conformational space adopted by a ubiquitin dimer. Here, molecular simulations on different levels of resolution can complement the structural information. We have combined exhaustive coarse grained and atomistic simulations of all eight possible ubiquitin dimers with a suitable dimensionality reduction technique and a new method to characterize protein-protein interfaces and the conformational landscape of protein conjugates. We found that ubiquitin dimers exhibit characteristic linkage type-dependent properties in solution, such as interface stability and the character of contacts between the subunits, which can be directly correlated with experimentally observed linkage-specific properties.
Post-translational modification of proteins by covalent attachment of ubiquitin is a key cellular process, regulating for example the fate and recycling of proteins. We present a new method to combine multiscale simulation with advanced analysis methods to characterize the states of ubiquitin-ubiquitin conjugates. We found that the linkage position affects the conformational space of ubiquitin dimers, determining the number and stability of relevant states, the character of subunit contacts and the nature of the surface exposed to possible binding partners.