Conformational and functional characterization of artificially conjugated non-canonical ubiquitin dimers

Abstract

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.

Machine Learning Driven Analysis of Large Scale Simulations Reveals Conformational Characteristics of Ubiquitin Chains

“…Such back-mapped structures can be used as input for atomistic MD simulation to validate the CG model prediction or for comparison to experimental data. 23,34 As described earlier, there is an intrinsic symmetry in the 2D map due to the arbitrary order of the two in principle indistinguishable Ub units in the RMD vector. This symmetry naturally reappears in the clustering.…”

“…Mean RMSD values for 3xUb clusters are obviously larger than for 2xUb configurations but are in the same range if normalized to the number of atoms, which were considered for the RMSD calculation (Figure S8.1, 2xUb: 0.002−0.006 nm; 3xUb: 0.002−0.010 nm). However, some clusters which are positioned next to each other in 2D are relatively different from a conformational point of view (compare cluster ids 11 with 25,12,34). Note that while the EncoderMap algorithm due to the multidimensional scaling like the distance cost aims for the preservation of distances (i.e., structural similarities) upon reducing the dimensionality of the RMD data to 2D, we expect that the network introduces certain distortions of the configutational space upon projection.…”

“…For a detailed description on representative structure determination and back-mapping see the Methods section. Such back-mapped structures can be used as input for atomistic MD simulation to validate the CG model prediction or for comparison to experimental data. , …”

“…This provided valuable insight into how the chain topology affects the accessibility of the Ub surface which has implications for linkage specific interactions. Moreover, the data could be directly correlated with and compared to chemical shift differences from NMR experiments. , Thus, NMR and simulation data could be combined to provide a bundle of structures that describes the conformational ensemble of differently linked Ub dimers in solution. A preliminary analysis of the solvent accessible surface of triUb indicates that the accessibility of the residues (and possibly of interaction patches) is dependent not only on the linkage but also on the position of the moiety in the chain and in consequence on the chain length.…”

Segmental and site-specific isotope labelling strategies for structural analysis of posttranslationally modified proteins

“…Segmental labelling increased the resolution of NMR signals of each individual Ub subunit, which would not have been possible by uniform labelling of the complete diUb due to multiple overlapping signals. However, the application of this labelling strategy is restricted to systems, such as that by Schneider et al,155 in which a disadvantageous influence of the non-native linkage can be excluded.Several strategies generating non-native linkages have also been applied for the conjugation of Ub to substrate proteins. For example, Ub attachment via disulfide linkages was described first by Chatterjee et al and Chen et al in 2010 157,158.…”