NMR spectroscopy of ubiquitin chains : Linkage chemistry and chain behavior

Abstract

As one of the key signaling processes in eukaryotic cells the fate of a protein is determined by its posttranslational modification with the small polypeptide ubiquitin. This is facilitated in a reversible manner by an enzymatic machinery forming a covalent isopeptide bond between the C-terminus of ubiquitin and lysine side chains of the protein. As this also happens at one of the seven lysine residues (K6, K11, K27, K29, K33, K48 and K63) or the N-terminus (M1) of the ubiquitin molecule itself, polymeric chains can be assembled consisting of multiple domains. Depending on length and linkage composition a multitude of chain architectures are achievable potentially invoking distinct cellular outcomes due to the interaction with respective downstream effector proteins. However, there is a persistent lack of knowledge about the appearance of the underlying conformational ensembles that are ultimately responsible for specific chain recognition. In this thesis this issue was addressed by applying high-resolution NMR spectroscopy to achieve a comprehensive characterization of diubiquitins representing the shortest kind of a ubiquitin chain. With respect to these diubiquitins the domain providing the lysine residue for conjugation is termed proximal and the domain providing the C-terminus is called distal. Note that the analysis of such multi-domain proteins connected via flexible linkers is a persistent challenge in modern structural biology, especially because of extensive interdomain dynamics disparaging the significance of common single structure representations. For that reason, efforts were also made to gain methodological advance in this regard including the integration of MD simulations. To get access to NMR spectroscopy an approach based on bioorthogonal click chemistry was established yielding diubiquitins with domain-specific isotope labeling of either one of both subunits that were artificially connected by a non-hydrolyzable linker. This allowed to perform a detailed structural, dynamical and functional analysis of diubiquitins comprising all linkage types apart from that referring to M1. A chemical shift perturbation analysis revealed that diubiquitins differ in the character of the interdomain interface and structural features of their proximal subunits. Furthermore, protein dynamics were probed on different time scales using 15N spin relaxation and amide proton exchange measurements, respectively. Intertwining the results with MD simulations gave an impression on the appearance of the conformational ensembles and the three-dimensional arrangement of the domains. Notably, all diubiquitins display a strong conformational heterogeneity that is less pronounced in case of K6- compared to K33-linked diubiquitin as additionally evidenced by an approach based on solvent paramagnetic relaxation enhancement that was developed here deliberately for illuminating this aspect. Moreover, the binding properties of diubiquitins were determined by NMR interaction studies providing support to the notion that the artificial linker used in this thesis for dimer formation in fact represents a reliable surrogate for the native isopeptide bond. This opened the door for functional studies unraveling the linkage-specific binding preferences of the deubiquitinating enzyme OTUD3. This revealed that the catalytic domain of the enzyme distinguishes K6- and K11-linked diubiquitin by the recognition of the proximal subunit and that it prefers the relaxed state of S65 phosphorylated ubiquitin over the retracted state for interaction. Note that apart from diubiquitin this thesis additionally includes the results of several NMR experiments that were performed to characterize site-specifically lysine acetylated ubiquitin and S65 phosphorylated NEDD8. The structural consequences of the respective posttranslational modifications were successfully brought into accordance with the results apparent from biochemical studies.