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Abstract

Ubiquitylation is an essential post-translational modification in eukaryotes. The reaction pathway requires at least three enzymes: a ubiquitin-activating enzyme (E1), a ubiquitin-conjugating enzyme (E2) and a ubiquitin-protein ligase (E3). E1 activates the C-terminal glycine residue (G76) of ubiquitin in an ATP-dependent reaction and later forms a thioester linkage through an active-site cysteine. Ubiquitin is then passed on to a conserved active-site cysteine of the E2 through a transesterification reaction. In case of the HECT E3 ubiquitin ligases a second transesterification step occurs and ubiquitin gets transferred from E2 to an active-site cysteine residue of the E3 HECT domain. Eventually HECT E3 ligases attach ubiquitin to a lysine of target proteins. Thereby, an isopeptide bond between G76 and the target lysine residue is formed. Structural studies of ubiquitylation intermediates are hampered by the intrinsic instability of all the thioester compounds. Thioesters are prone to hydrolysation rendering them inaccessible to NMR studies or crystallization assays. In order to characterize the structure of a ubiquitin thioester with a HECT domain of an E3 enzyme, we overcome the instability of the thioester by forming disulfide compounds between a C-terminal mutant of ubiquitin (G76C) and the catalytic cysteine of the HECT domain. The disulfide linkage is stable and mimics the bond distances of thioester bonds. We show here that we can form these Ubiquitin-HECT domain disulfides under non-reducing condition and that we can purify them to homogeneity. Furthermore, we present initial NMR spectra of a Ubiquitin-HECT domain disulfide providing the first structural insight into a HECT reaction intermediate in solution.