Altered cofactor regulation with disease-associated p97/VCP mutations

Abstract

Dominant mutations in p97/VCP (valosin-containing protein) cause a rare multisystem degenerative disease with varied phenotypes that include inclusion body myopathy, Paget's disease of bone, frontotemporal dementia, and amyotrophic lateral sclerosis. p97 disease mutants have altered N-domain conformations, elevated ATPase activity, and altered cofactor association. We have now discovered a previously unidentified disease-relevant functional property of p97 by identifying how the cofactors p37 and p47 regulate p97 ATPase activity. We define p37 as, to our knowledge, the first known p97-activating cofactor, which enhances the catalytic efficiency (k cat /K m ) of p97 by 11-fold. Whereas both p37 and p47 decrease the K m of ATP in p97, p37 increases the k cat of p97. In contrast, regulation by p47 is biphasic, with decreased k cat at low levels but increased k cat at higher levels. By deleting a region of p47 that lacks homology to p37 (amino acids 69-92), we changed p47 from an inhibitory cofactor to an activating cofactor, similar to p37. Our data suggest that cofactors regulate p97 ATPase activity by binding to the N domain. Induced conformation changes affect ADP/ATP binding at the D1 domain, which in turn controls ATPase cycling. Most importantly, we found that the D2 domain of disease mutants failed to be activated by p37 or p47. Our results show that cofactors play a critical role in controlling p97 ATPase activity, and suggest that lack of cofactorregulated communication may contribute to p97-associated disease pathogenesis.AAA ATPase | p97/VCP | MSP1 | p47 | steady-state kinetics

Valosin-containing protein (VCP)–Adaptor Interactions are Exceptionally Dynamic and Subject to Differential Modulation by a VCP Inhibitor

“…8C and Table I). This affinity is ϳ10-fold tighter than what was reported from isothermal titration calorimetry studies (52,53), but is close to the affinity measured in a pair of surface plasmon resonance (SPR) studies (20 -31 nM) (50,54). However, there are significant problems with measuring NSFL1C-VCP interactions by SPR because of the oligomeric nature of both proteins.…”

Control of p97 function by cofactor binding

“…In contrast, the inhibitory effect of p47 might be the consequence of promoting the ADP-locked state that prevents ATP binding as described for the wild-type protein (see above). Taken together, the results of Zhang et al for both wild-type and mutant proteins show that cofactors play a critical role in controlling p97 ATPase activity, and suggest that a lack of cofactor-regulated communication may contribute to p97-associated disease pathogenesis [112].…”

“…In contrast, p37 binding to the N domain most likely induces conformational changes which convert the D1 domain into the ADP-open state that allows ATP binding to D1, which in turn activates the D2 domain. These findings suggest that distinct cofactors can induce different conformational changes in N-D1 that regulate ADP/ATP binding to the D1 domain, which in turn controls both the D1 and D2 ATPase cycles [112].…”

“…Recently, the link between the regulation of p97 ATPase activity by cofactors has been studied in more detail for p37 and p47 [112]. Zhang et al identified a region of about 25 unstructured amino acids in p47 that is not involved in p97 binding and is missing in p37, which is responsible for the inhibitory effect of p47.…”

“…Interestingly, the decreased binding to UBXD1 is associated with a blockage of the endolysosomal trafficking of caveolin 1 [10]. Although disease mutants and wild-type p97 interact with both cofactors in a similar manner in vitro, disease mutants no longer can be activated by p37, but are still inhibited by p47 [112]. In disease mutants the described conformational changes for the wild-type protein (see above) are already induced, as they are in the active ADP-open state, which might explain why no additional activation can be observed in these mutants with p37.…”