Allosteric Regulation of Nucleotide Binding to the Proteasomal ATPases

Abstract

Protein degradation by the eukaryotic 26S proteasome or the homologous archaeal PAN-20S proteasome complex is a multistep process that requires ATP hydrolysis by the proteasome-associated AAA ATPase complex. However, the mechanisms by which these hexameric ATPases bind and hydrolyze ATP to promote protein breakdown are poorly understood. Although PAN contains six identical ATPase subunits, we found that it exhibits three types of ATP binding sites: 2 high affinity conformations (ATP-binding sites), 2 with lower affinity (ADP-binding sites), and 2 with conformations that do not bind nucleotides. Correlation of ADP off rates with rates of ATP hydrolysis suggests that ADP leaving may be the rate limiting step in hydrolysis. ATP binding to the high and lower affinity sites has distinct functional consequences on the proteasome. With two ATPγS molecules bound, PAN maximally stimulates opening of the gated channel for substrate entry into the 20S proteasome and has a high affinity for the 20S. However, the binding of 4 ATPγS reduces PAN’s affinity for the 20S, which can be explained by steric hindrances in the PAN-20S interface. Because ATP binding drives the association of the C-termini of the ATPase with the 20S and only two ATPase subunits bind ATP for maximal function it’s likely that only two ATPases’ C-termini dock into the 20S at any time and in a predictable pattern mirroring the cyclical pattern of ATP hydrolysis. This observation suggests how the symmetrically mismatched hexameric ATPase ring associates with the heptameric 20S proteasome to regulate substrate degradation.