Functional Consequences of Nucleotide Binding to the Proteasomal ATPases

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 utilize ATP to promote protein breakdown and activate proteasomal function are poorly understood. Although PAN contains six identical ATPase subunits, we found that it exhibits three types of binding sites: 2 high affinity conformations (Kd=0.2uM), 2 with lower affinity (Kd=60uM), and 2 with conformations that fail to bind ATP. In fact, PAN never bound more than four of any type of nucleotide (ADP, ATP, or the nonhydroylyzable analog ATPγS), even at high concentrations. 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 as well as for protein substrate. However, the binding of 4 ATPγS reduces PAN's ability to stimulate gate opening as well as its affinity for the 20S and substrates. These functional consequences of nucleotide binding are conserved in the mammalian 26S proteasome, since gate opening exhibited nearly identical multiphasic dependence on ATPγS concentration as found for the PAN‐20S complex. 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. This observation suggests how the hexameric ATPase ring associates with the heptameric 20S proteasome to regulate substrate degradation.