The p97 protein is a highly conserved, abundant, and structurally dynamic homohexameric AAA enzyme. Monomers contain N, D1, and D2 domains. A truncated p97 protein containing the N and D1 domains and the D1-D2 linker (ND1L) exhibits 79% of WT activity. To investigate the effect of the linker specifically on the D1 domain, we demonstrated that the flexible linker region is essential for D1 activity. Conserved leucine 464 in the D1-D2 linker region is critical for degradation of two p97 substrates. Here, we show that leucine 464 is important for p97 regulation by cofactors p37, p47, and the Npl4-Ufd1 heterodimer (NU). Changing leucine to alanine (L464A), proline (L464P), or glutamate (L464E) altered p47-regulated ATPase activities without significant effects on basal ATPase activity. Steady-state kinetic analysis showed that p47 increased the maximum rate of ATP turnover (k<sub>cat</sub>) for L464A, L464P, and L464E mutants of p97 but not for WT. An L464P mutant of ND1L reduced D1 ATPase activity by 116-fold. However, p37 and p47 raised the kcat of L464P-ND1L, suggesting that they induce linker conformations that facilitate ATP hydrolysis. NU inhibited D1 ATPase activities of WT-ND1L but activated full-length p97. In addition, a p97 disease mutation, R155H, exhibits high D2 ATPase activity but L464E blocked this activation and eliminated p47-inhibition of R155H p97. Thus, the linker helps D1 maintain a competent conformation and relays cofactor binding effects from the N-domain to the D1 and D2 ATPase domains (~50Å away).<br/>

Cofactor regulation is important for the function of p97/VCP AAA ATPase

254th National Meeting (2017)

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