Structure and Catalytic Regulatory Function of Ubiquitin Specific Protease 11 N-Terminal and Ubiquitin-like Domains

Abstract

The ubiquitin specific protease 11 (USP11) is implicated in DNA repair, viral RNA replication, and TGFβ signaling. We report the first characterization of the USP11 domain architecture and its role in regulating the enzymatic activity. USP11 consists of an N-terminal “domain present in USPs” (DUSP) and “ubiquitin-like” (UBL) domain, together referred to as DU domains, and the catalytic domain harboring a second UBL domain. Crystal structures of the DU domains show a tandem arrangement with a shortened β-hairpin at the two-domain interface and altered surface characteristics compared to the homologues USP4 and USP15. A conserved VEVY motif is a signature feature at the two-domain interface that shapes a potential protein interaction site. Small angle X-ray scattering and gel filtration experiments are consistent with the USP11DU domains and full-length USP11 being monomeric. Unexpectedly, we reveal, through kinetic assays of a series of deletion mutants, that the catalytic activity of USP11 is not regulated through intramolecular autoinhibition or activation by the N-terminal DU or UBL domains. Moreover, ubiquitin chain cleavage assays with all eight linkages reveal a preference for Lys63-, Lys6-, Lys33-, and Lys11-linked chains over Lys27-, Lys29-, and Lys48-linked and linear chains consistent with USP11’s function in DNA repair pathways that is mediated by the protease domain. Our data support a model whereby USP11 domains outside the catalytic core domain serve as protein interaction or trafficking modules rather than a direct regulatory function of the proteolytic activity. This highlights the diversity of USPs in substrate recognition and regulation of ubiquitin deconjugation.

PJA2 ubiquitinates the HIV-1 Tat protein with atypical chain linkages to activate viral transcription

“…One of these, PPM1G, has recently been shown to dephosphorylate CDK9 to locally disassemble the 7SK snRNP at the HIV promoter12, supporting that the screen can readily identify functionally important Tat host factors. Examining the functions of these nine positives revealed an unexpected enrichment for ubiquitin signaling proteins, with three E3 ligases (ZFP91, PJA2, and UBE2O)3738394041, two substrate receptors for multi-subunit E3 ligases (DCAF16, FBX3)4243, and one de-ubiquitinating enzyme (USP11)44 (Fig. 1c).…”

Structural Basis of the Recruitment of Ubiquitin-specific Protease USP15 by Spliceosome Recycling Factor SART3

“…Domain Swapping and Hinge Residues-Several previous studies (18,(27)(28)(29) have analyzed the structures and dimerization of the DUSP-UBL domain of USP4, USP11, and USP15 in detail and identified residues on the DU finger and the DUSP domain to be the main cause of different oligomerization mechanisms of the USPs. USP4 exists as a dimer in the solution and in the crystal lattice.…”

“…Previous studies (18,(27)(28)(29) established that the USP4 DUSP-UBL domain forms a dimer in solution and in the crystal. Detailed structural analysis revealed the DU finger of one USP4 molecule interacts with a hydrophobic patch on the DUSP domain of the other molecule (Fig.…”

Recent Advances in the Discovery of Deubiquitinating Enzyme Inhibitors

“…Structural information on how Ubls and additional domains of DUBs interact continues to emerge [38]. This is demonstrated by recent publications on the first characterisation of USP11 domain architecture and the discovery of a new binding site on USP7 (via the second of its five Ubls) [39,40]. The presence of additional domains, as well as cofactors for a few USPs [41,42], in theory provides multiple allosteric options for DUB inhibition.…”