A conserved phenylalanine reveals a regulatory loop within p97 AAA ATPase

Proteolytic machineries containing multi-subunit protease complexes and AAA-ATPases, play a key role in protein quality control and protein homeostasis. The proteasome is the most prominent regulated protease complex and is controlled by an AAA-ATPases ring. In this complex, nucleotide binding and hydrolysis regulate multiple aspects of substrate recognition, unfolding, de-ubiquitination and translocation into the 20S catalytic particle.

We revealed a molecular mechanism that supports the coordination between the archaeal regulatory ATPase (PAN) and the 20S proteasome, which relies on the conformation of a specific phenylalanine residue, conserved in all other AAA ATPases as well.

To demonstrate the generality of this mechanism we mutated the corresponding phenylalanine residue in the eukaryotic AAA ATPase complex, p97. The biochemical properties of p97 were altered in a similar manner as did PAN ATPase. Furthermore, Cryo-EM structures of the phenylalanine mutant indicated that p97 was trapped at a specific conformation identical to that of the ATP bound state of both ATPase rings regardless of the presence of nucleotide.

This stabilized mutant allowed us to capture new potential functional partners of p97 as well as to reveal new ATP binding and hydrolysis dependent steps in the function of p97.