Naturally and synthetically linked lys48 diubiquitin: a QM/MM study

Abstract

Site-specific chemical conjugates of proteins with ubiquitin-like modifiers are a powerful tool for biochemists to probe the function of these post-translational modifications. As a prominent example, the seven native isopeptide-linked diubiquitins serve as models to study polyubiquitin chains, but not all of them can be prepared by enzymatic means. To date, several approaches to access synthetically conjugated diubiquitins have been reported, however, an experimental structure of such a molecule with an unnatural linker remains elusive. Therefore, here we present structural models of the Lys48-connected diubiquitin with the natural isopeptide and a synthetic triazole linker using hybrid quantum mechanical and molecular mechanical (QM/MM) methods. Despite differences in the flexibility of the linkers, the systems with the synthetic and natural linker were found to be very similar in regard to the analyzed geometric features. This provides computational support for the use of the synthetically linked conjugate as a mimic of the natural isopeptide bonded diubiquitin.

Comparison of native and non-native ubiquitin oligomers reveals analogous structures and reactivities

“…To be recognized and processed by DUBs, Ub conjugates bearing non‐native linkages should recapitulate native structural features. The conformations and flexibility of select non‐native Ub linkages have previously been evaluated with computational methods . However, to effectively address whether non‐native linkages can adopt native chain conformations and functions, a solution‐based characterization is needed.…”

Peeling away the layers of ubiquitin signaling complexities with synthetic ubiquitin–protein conjugates

“…Calculations using Rosetta, for example, revealed that a disulfide-linked Ub conjugate samples the same conformational space as the native conjugate [35]. On the other hand, molecular dynamics simulations of Ub dimers bearing a triazole linker showed the artificial linker renders the conjugate less flexible compared to the native dimer [36]. These results suggest that the disulfide linkage might not affect the on/off rates of binding interactions while the triazole linker could introduce some deviation.…”

Dissecting Ubiquitin Signaling with Linkage‐Defined and Protease Resistant Ubiquitin Chains

“…We next investigated whether connecting the individual ubiquitin moieties with triazoles still results in chains with native-like properties, as it has already been demonstrated for di-ubiquitin by Mootz and co-workers. [20] For this purpose, an antibody that selectively binds K48-linked ubiquitin chains was employed ( Figure S2). Indeed, K48-linked ubiquitin oligomers were detected while no signals could be observed for the other chain linkages.…”