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Abstract

HECT-type ligases are large monomeric proteins that covalently attach ubiquitin (Ub) to Lys residues in substrates. Ubiquitination is a key regulatory mechanism in signal transduction and one of the structurally most complex post-translational modifications. Ub can be conjugated to its targets as a single moiety at one or multiple sites or as a poly-Ub chain that can be linked via any of the ven Lys residues in Ub or via the Ub N-terminus. This vast array of Ub modifications creates distinct cellular signals that control virtually all signal transduction pathways in eukaryotes. To explore how HECT-type Ub ligases elongate Ub chains we have used solution-state NMR spectroscopy in combination with biochemical assays. Using our recently developed Met scanning approach we have determined key residues for non-covalent Ub binding on HECT domains. Furthermore, we have performed chemical shift perturbation studies using short Ub chains where we have specifally labeled individual Ub moeties. Our results provide important insights into the mechanism of Ub chain elongation and enzyme processivity in HECT-type Ub ligases.