The 26S proteasome is an essential molecular machine for specific protein degradation in eukaryotic cells.1 The 26S proteasome is formed by a central 20S core particle (CP) capped by two 19S regulatory particle (RP) at both ends. The Rpn9 protein is a non-ATPase subunit located in the lid complex of the 19S RP, and is identified to be essential for the integrity and efficient assembly the 26S proteasome. Bioinformatics analyses of Rpn9 indicated that it contains an N-terminal domain (NTD) and a C-terminal proteasome-cyclosome-initiation factor (PCI) domain. A structure model of the 26S proteasome derived from cryo-EM maps suggested that the NTD and PCI domains of Rpn9 may separately contribute to interactions with the Rpn10 and Rpn5 subunits in the 19S RP.2 However, high-resolution structures of Rpn9 and the details of its interactions are still lacking.
Herein we present solution NMR studies of the 46 kDa Saccharomyces cerevisiae Rpn9 protein. The structures of the individual domains of Rpn9 as well as the full-length protein are determined. Similar to the PCI domain of the CSN7 subunit of the COP9 signalosome, the C-terminal PCI domain of yeast Rpn9 is comprised of a helix bundle and a winged-helix subdomain. The NTD domain of Rpn9 is comprised of an anti-parallel helical solenoid that resembles the tetratricopeptide repeats (TPR)-like fold. In the full-length protein, a three-helice bundle formed by the linker region connects the two domains together. Fitting of the full-length Rpn9 structure in the cryo-EM map of the 26S proteasome reveals its possible contact surface with Rpn5 and Rpn10 subunits. NMR titration experiments in combination with biochemical and mutagenesis investigation verify the interaction patterns, and reveal essential residues and structural motifs that contribute to the interactions.