Final Report Summary - UBLIGATION (Structural Basis of Ubiquitin Protein Ligation)
Ubiquitination is one of the most abundant and structurally most complex post-translational modifications. Ubiquitin (Ub) may be attached to substrates as a monomer or as poly-Ub chains. The latter can be linked via any of the seven Ub Lys residues or via the Ub N-terminus. The type of Ub chain dictates the cellular fate of the substrate, e.g. degradation or endocytosis. An enzymatic cascade of an activating (E1), a conjugating (E2), and a ligating (E3) enzyme catalyzes Ub attachment to substrate Lys residues. Ub ligases dictate target specificity as they catalyze the formation of the ultimate substrate-Ub adduct. To prevent premature ubiquitination, E3 activity is tightly controlled, and deregulation of E3s causes various human diseases including cancer. E3s are therefore promising lead targets for drug design.
HECT-type E3s contain a catalytic Cys that accepts Ub from the E2~Ub intermediate to form an E3~Ub thioester prior to transferring Ub to the substrate. HECT domains comprise ~350 aa and have a bilobal structure with the N-terminal (N-) lobe containing the E2-binding surface and the C-terminal (C-) lobe bearing the catalytic Cys. A flexible hinge region between the two lobes allows the C-lobe to adopt different orientations relative to the N-lobe. This suggests that HECT domains undergo large conformational changes during Ub transfer.
C2-WW-HECT (Nedd4) E3s form a major group among HECT-type ligases. Nedd4 E3s regulate the trafficking and stability of signaling proteins through mono- and K63-linked poly-ubiquitination. Recent structural studies support a sequential Ub addition mechanism for Nedd4 HECT domains where a non-covalent Ub-binding surface on the N-lobe holds the Ub moiety on the substrate in place to promote chain elongation (Ogunjimi AA, Wiesner S et al., JBC, 2010). Although the details of the catalytic mechanisms underlying HECT-mediated Ub transfer are beginning to emerge, important questions as to the regulation of HECT domain activity, the dynamics underlying HECT-mediated Ub transfer and the mode of substrate recognition remain to be answered.
Over the duration of this grant, work in my lab has focused on the mechanisms underlying the regulation and substrate recognition of HECT-type E3s. Since the poor stability of HECT domains precluded traditional H,N-based NMR studies, we have developed an NMR method termed methionine scanning (Stoffregen et al, Structure, 2012). This method has enabled us to study HECT domain interactions and conformational changes by methyl NMR with per-residue resolution.
Using our Met scanning approach together with functional assays, we have investigated the structural details of the intramolecular interaction between the C2 and the catalytic HECT domain that inhibits the activity of Nedd4-family E3s. We have identified a dual autoinhibition mechanism where the C2 domain induces an inactive HECT domain conformation and at the same time blocks non-covalent Ub binding and thereby inhibits enzyme processivity (Mari et al, Structure, 2014).
Furthermore, we have developed a protocol for the efficient production of HECT-Ub disulfide linkages to mimic the hydrolytically unstable HECT~Ub thioester. This has enabled us to solve the crystal structure of a HECT~Ub thioester and has allowed us to gain insight into the mechanism of Ub transfer from the E2 to the HECT domain.
To gain insight into E3:substrate recognition, we have studied substrates of Smurf Ub ligases involved in cell polarity such as Par-3. We have mapped the binding surface of the Smurf2 HECT domain on the Par-3 PDZ3 domain providing the first structural insight into a HECT:substrate interaction. Furthermore, we have undertaken a structural characterization of di-Ub binding to HECT domains, as a minimal unit of to investigate how a poly-Ub chain may be anchored close to the catalytic site of the HECT domain. Taken together, our results suggest a model for E3 regulation where the C2-mediated autoinhibition can be released by substrate binding. Once the substrate is mono-ubiquitinated, the Ub moiety replaces the substrate on the HECT domain to promote Ub-chain elongation.
http://www.eb.tuebingen.mpg.de/research-groups/silke-wiesner.html
HECT-type E3s contain a catalytic Cys that accepts Ub from the E2~Ub intermediate to form an E3~Ub thioester prior to transferring Ub to the substrate. HECT domains comprise ~350 aa and have a bilobal structure with the N-terminal (N-) lobe containing the E2-binding surface and the C-terminal (C-) lobe bearing the catalytic Cys. A flexible hinge region between the two lobes allows the C-lobe to adopt different orientations relative to the N-lobe. This suggests that HECT domains undergo large conformational changes during Ub transfer.
C2-WW-HECT (Nedd4) E3s form a major group among HECT-type ligases. Nedd4 E3s regulate the trafficking and stability of signaling proteins through mono- and K63-linked poly-ubiquitination. Recent structural studies support a sequential Ub addition mechanism for Nedd4 HECT domains where a non-covalent Ub-binding surface on the N-lobe holds the Ub moiety on the substrate in place to promote chain elongation (Ogunjimi AA, Wiesner S et al., JBC, 2010). Although the details of the catalytic mechanisms underlying HECT-mediated Ub transfer are beginning to emerge, important questions as to the regulation of HECT domain activity, the dynamics underlying HECT-mediated Ub transfer and the mode of substrate recognition remain to be answered.
Over the duration of this grant, work in my lab has focused on the mechanisms underlying the regulation and substrate recognition of HECT-type E3s. Since the poor stability of HECT domains precluded traditional H,N-based NMR studies, we have developed an NMR method termed methionine scanning (Stoffregen et al, Structure, 2012). This method has enabled us to study HECT domain interactions and conformational changes by methyl NMR with per-residue resolution.
Using our Met scanning approach together with functional assays, we have investigated the structural details of the intramolecular interaction between the C2 and the catalytic HECT domain that inhibits the activity of Nedd4-family E3s. We have identified a dual autoinhibition mechanism where the C2 domain induces an inactive HECT domain conformation and at the same time blocks non-covalent Ub binding and thereby inhibits enzyme processivity (Mari et al, Structure, 2014).
Furthermore, we have developed a protocol for the efficient production of HECT-Ub disulfide linkages to mimic the hydrolytically unstable HECT~Ub thioester. This has enabled us to solve the crystal structure of a HECT~Ub thioester and has allowed us to gain insight into the mechanism of Ub transfer from the E2 to the HECT domain.
To gain insight into E3:substrate recognition, we have studied substrates of Smurf Ub ligases involved in cell polarity such as Par-3. We have mapped the binding surface of the Smurf2 HECT domain on the Par-3 PDZ3 domain providing the first structural insight into a HECT:substrate interaction. Furthermore, we have undertaken a structural characterization of di-Ub binding to HECT domains, as a minimal unit of to investigate how a poly-Ub chain may be anchored close to the catalytic site of the HECT domain. Taken together, our results suggest a model for E3 regulation where the C2-mediated autoinhibition can be released by substrate binding. Once the substrate is mono-ubiquitinated, the Ub moiety replaces the substrate on the HECT domain to promote Ub-chain elongation.
http://www.eb.tuebingen.mpg.de/research-groups/silke-wiesner.html