Dynamic flexibility of p97 AAA ATPase is important for cofactor regulation

The p97 protein is a highly conserved, abundant, functionally diverse, and structurally dynamic AAA ATPase. We have previously shown that regulation of p97's ATPase activity by its cofactors p37 and p47 is important for its normal cellular role. The conserved linker residue Leu‐464 has been reported to be critical for degradation of one p97 substrate in cells; structural data suggests that Leu‐464 controls interprotomer transmission. Here, we demonstrate that Leu‐464 controls communication of cofactor binding from the N‐domain to the D1 and D2 ATPase domains by affecting the regulation of three p97 cofactors: p37, p47, and the Npl4‐Ufd1 heterodimer (NU). Leu‐464 mutants abolished p47's ATPase‐inhibitory effect and produced activation at higher p47 concentrations. Both p37‐ and NU‐mediated activation decreased slightly for L464A and L464E, but increased for L464P. Using ND1L protein, we demonstrate that the flexible linker region is essential for the D1 ATPase activity: a point mutation, L464P, reduced ND1L activity to 2%. We demonstrate that all three cofactors decrease the K m of ATP to ND1L. NU inhibited D1 activity (k cat ) for WT‐, L464E‐, and L464P‐ND1L proteins. Neither p37 nor p47 affected the k cat values for WT‐ and L464E‐ND1L, but both raised the k cat of L464P‐ND1L (14‐fold and 7‐fold, respectively). This significant increase suggests that p37 or p47 binding induces conformational changes in the linker region to correct the defect caused by the Pro residue. Most importantly, we found that L464E blocked the activation of the disease variant R155H and also completely disrupted the p47‐mediated inhibition of R155H. The linker region not only assists D1 in maintaining a competent conformation, but also transmits the binding effect of p97 cofactors from the N‐domain (44.5 Å away) to the D1 and D2 domains.