Details

Autor(en) / Beteiligte

• Aufderheide, Antje
• Florian Beck
• Florian Stengel
• Michaela Hartwig
• Andreas Schweitzer
• Günter Pfeifer
• Alfred L. Goldberg
• Eri Sakata
• Wolfgang Baumeister
• Friedrich Füörster

Titel

Structural characterization of the interaction of Ubp6 with the 26S proteasome

Ist Teil von

• Proceedings of the National Academy of Sciences - PNAS, 2015-07, Vol.112 (28), p.8626-8631

Ort / Verlag

United States: National Academy of Sciences

Erscheinungsjahr

2015

Link zum Volltext

Quelle

MEDLINE

Beschreibungen/Notizen

• In eukaryotic cells the 26S proteasome is responsible for the regulated degradation of intracellular proteins. The function of this large macromolecular machine is regulated by many cofactors, most notably the deubiquitylating enzyme ubiquitin C-terminal hydrolase 6 (Ubp6). Here, we investigate the structure of Ubp6 bound to the 26S proteasome and explore its influence on the conformational landscape of the 26S proteasome. Our structure reveals that Ubp6’s active site may contribute to a large composite active site, also formed by the mouth of the proteasomal ATPase ring and the active site of deubiquitylating enzyme regulatory particle non-ATPase 11. Moreover, Ubp6 modulates the conformational landscape of the proteasome, favoring an intermediate state, which may explain the effects of Ubp6 on proteasomal degradation. In eukaryotic cells, the 26S proteasome is responsible for the regulated degradation of intracellular proteins. Several cofactors interact transiently with this large macromolecular machine and modulate its function. The deubiquitylating enzyme ubiquitin C-terminal hydrolase 6 [Ubp6; ubiquitin-specific protease (USP) 14 in mammals] is the most abundant proteasome-interacting protein and has multiple roles in regulating proteasome function. Here, we investigate the structural basis of the interaction between Ubp6 and the 26S proteasome in the presence and absence of the inhibitor ubiquitin aldehyde. To this end we have used single-particle electron cryomicroscopy in combination with cross-linking and mass spectrometry. Ubp6 binds to the regulatory particle non-ATPase (Rpn) 1 via its N-terminal ubiquitin-like domain, whereas its catalytic USP domain is positioned variably. Addition of ubiquitin aldehyde stabilizes the binding of the USP domain in a position where it bridges the proteasome subunits Rpn1 and the regulatory particle triple-A ATPase (Rpt) 1. The USP domain binds to Rpt1 in the immediate vicinity of the Ubp6 active site, which may effect its activation. The catalytic triad is positioned in proximity to the mouth of the ATPase module and to the deubiquitylating enzyme Rpn11, strongly implying their functional linkage. On the proteasome side, binding of Ubp6 favors conformational switching of the 26S proteasome into an intermediate-energy conformational state, in particular upon the addition of ubiquitin aldehyde. This modulation of the conformational space of the 26S proteasome by Ubp6 explains the effects of Ubp6 on the kinetics of proteasomal degradation.