A Role for the Proteasome Alpha2 Subunit N-Tail in Substrate Processing

The proteolytic active sites of the 26S proteasome are sequestered within the catalytic chamber of its 20S core particle (CP). Access to this chamber is through a narrow channel defined by the seven outer alpha subunits. In the resting state, the N-termini of neighboring alpha subunits form a gate blocking access to the channel. The attachment of the activators or regulatory particles rearranges the blocking alpha subunit N-termini facilitating the entry of substrates. By truncating or mutating each of the participating alpha N-termini, we report that whereas only a few N-termini are important for maintaining the closed gate, all seven N-termini participate in the open gate. Specifically, the open state is stabilized by a hydrogen bond between an invariant tyrosine (Y) in each subunit with a conserved aspartate (D) in its counterclockwise neighbor. The lone exception is the alpha 1-alpha 2 pair leaving a gap in the ring circumference. The third residue (X) of this YD(X) motif aligns with the open channel. Phenylalanine at this position in the alpha 2 subunit comes in direct contact with the translocating substrate. Consequently, deletion of the alpha 2 N-terminal tail attenuates proteolysis despite the appearance of an open gate state. In summary, the interlacing N-terminal YD(X) motifs regulate both the gating and translocation of the substrate.

Automated summary

The active sites of the 26S proteasome are located in the catalytic chamber of its 20S core particle. Access to this chamber is through a narrow channel formed by the seven outer alpha subunits. The gate blocking this channel is formed by the N-termini of neighboring alpha subunits, and attachment of activators rearranges these N-termini, allowing entry of substrates. A hydrogen bond between an invariant tyrosine (Y) in each subunit and a conserved aspartate (D) in its counterclockwise neighbor stabilizes the open state, except for the alpha 1-alpha 2 pair which leaves a gap in the ring. The third residue (X) of the YD(X) motif aligns with the open channel, and deletion of the alpha 2 N-terminal tail attenuates proteolysis despite the appearance of an open gate state.