Mechanism of Gate Opening in the 20S Proteasome by the Proteasomal ATPases

Abstract

Substrates enter the cylindrical 20S proteasome through a gated channel that is regulated by the ATPases in the 19S regulatory particle in eukaryotes or the homologous PAN ATPase complex in archaea. These ATPases contain a conserved C-terminal hydrophobic-tyrosine-X (HbYX) motif that triggers gate opening upon ATP binding. Using cryo-electron microscopy, we identified the sites in the archaeal 20S where PAN's C-terminal residues bind and determined the structures of the gate in its closed and open forms. Peptides containing the HbYX motif bind to 20S in the pockets between neighboring alpha subunits where they interact with conserved residues required for gate opening. This interaction induces a rotation in the alpha subunits and displacement of a reverse-turn loop that stabilizes the open-gate conformation. This mechanism differs from that of PA26/28, which lacks the HbYX motif and does not cause alpha subunit rotation. These findings demonstrated how the ATPases' C termini function to facilitate substrate entry.

Structural Insights into the Conformational Diversity of ClpP from Bacillus subtilis

“…Furthermore, it shares functional and architectural features with other energy-dependent protease systems such as HslVU in prokaryotes and the 26S proteasome in eukaryotes (Bochtler et al, 1999;Pickart and Cohen, 2004;Rohrwild et al, 1996;Song et al, 2000;Yu and Houry, 2007). Structural and biochemical studies of these ATPdependent proteases have provided a wealth of information regarding the mechanism of substrate recognition and protease activation Lee et al, 2010a;Park and Song, 2008;Park et al, 2007;Rabl et al, 2008;Ramachandran et al, 2002;Song et al, 2000;Sousa et al, 2000;Wang et al, 2001). HslU, ClpA/ClpX and the 19S regulatory cap utilize a domain(s) for recognizing substrate proteins and delivering these in an unfolded form, generated by ATP hydrolysis, to the matching proteases (Bochtler et al, 1999;Park and Song, 2008;Park et al, 2007;Sauer and Baker, 2011;Song and Eck, 2003).…”

“…HslU, ClpA/ClpX and the 19S regulatory cap utilize a domain(s) for recognizing substrate proteins and delivering these in an unfolded form, generated by ATP hydrolysis, to the matching proteases (Bochtler et al, 1999;Park and Song, 2008;Park et al, 2007;Sauer and Baker, 2011;Song and Eck, 2003). Structural studies of HslVU complex, ClpP in complex with activator acyldepsipeptide (ADEP), the 20S proteasome in complex with the 11S proteasome activator PA28, and the Cterminal peptide of proteasome-activating nucleosidase (PAN) have revealed the activation mechanism of the protease components (Lee et al, 2010a;Li et al, 2010;Rabl et al, 2008;Sousa et al, 2000;Stadtmueller and Hill, 2011;Whitby et al, 2000). As already mentioned, the cylindrical shape of the protease component has two substrate entry pores at both ends doubly capped by two AAA+ ATPases.…”

Keeping proteasomes under control—a role for phosphorylation in the nucleus

“…During protein degradation, the ATPases' C termini dock into pockets between the α-subunits and trigger opening of the gated channel for substrate entry (19). This gate is formed by the N termini of the α-subunits, and possibly phosphorylation influences gate opening and thus may contribute to the enhanced peptide hydrolysis noted earlier (20).…”

The proteasome: molecular machinery and pathophysiological roles

“…The Rpt ring is thought to pull substrates into the central pore of the Rpt ring with suffi cient force to promote unfolding of substrate structure domains and translocate them to the CP channel for degradation. Among the six Rpt subunits, only Rpt2, Rpt3, and Rpt5 contain the C-terminal hydrophobictyrosine-X (HbYX) motif, and the Rpt2 and Rpt5 subunits specifi cally facilitate the gate opening (Kohler et al , 2001 ;Smith et al , 2007 ;Gillette et al , 2008 ;Rabl et al , 2008 ).…”