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Cooperative subunit dynamics modulate p97 function

Significance Mutations in the highly conserved hexameric p97 enzyme are associated with a series of diseases affecting muscle, bone, and brain, and have been linked to changes to the orientation of the N-terminal domain (NTD) in each p97 subunit. Although studies have focused on p97 constructs where all protomers are WT or all contain a disease-causing mutation, a strong body of evidence suggests that p97 molecules are heterogeneous, containing a mixture of these subunits in patients. We have used solution NMR to study such heterohexameric structures, showing that the NTD conformation of a disease mutant can be somewhat remedied by WT neighbors, leading to a partial restoration of interactions with adaptor molecules. The mechanism of this allosteric process is investigated. p97 is an essential hexameric AAA+ ATPase involved in a wide range of cellular processes. Mutations in the enzyme are implicated in the etiology of an autosomal dominant neurological disease in which patients are heterozygous with respect to p97 alleles, containing one copy each of WT and disease-causing mutant genes, so that, in vivo, p97 molecules can be heterogeneous in subunit composition. Studies of p97 have, however, focused on homohexameric constructs, where protomers are either entirely WT or contain a disease-causing mutation, showing that for WT p97, the N-terminal domain (NTD) of each subunit can exist in either a down (ADP) or up (ATP) conformation. NMR studies establish that, in the ADP-bound state, the up/down NTD equilibrium shifts progressively toward the up conformation as a function of disease mutant severity. To understand NTD functional dynamics in biologically relevant p97 heterohexamers comprising both WT and disease-causing mutant subunits, we performed a methyl-transverse relaxation optimized spectroscopy (TROSY) NMR study on a series of constructs in which only one of the protomer types is NMR-labeled. Our results show positive cooperativity of NTD up/down equilibria between neighboring protomers, allowing us to define interprotomer pathways that mediate the allosteric communication between subunits. Notably, the perturbed up/down NTD equilibrium in mutant subunits is partially restored by neighboring WT protomers, as is the two-pronged binding of the UBXD1 adaptor that is affected in disease. This work highlights the plasticity of p97 and how subtle perturbations to its free-energy landscape lead to significant changes in NTD conformation and adaptor binding.