The role of the N-domain in the ATPase activity of the mammalian AAA ATPase p97/VCP

Abstract

p97/valosin-containing protein (VCP) is a type II ATPase associated with various cellular activities that forms a homohexamer with each protomer containing an N-terminal domain (N-domain); two ATPase domains, D1 and D2; and a disordered C-terminal region. Little is known about the role of the N-domain or the C-terminal region in the p97 ATPase cycle. In the p97-associated human disease inclusion body myopathy associated with Paget disease of bone and frontotemporal dementia, the majority of missense mutations are located at the N-domain D1 interface. Structure-based predictions suggest that such mutations affect the interaction of the N-domain with D1. Here we have tested ten major inclusion body myopathy associated with Paget disease of bone and frontotemporal dementia-linked mutants for ATPase activity and found that all have increased activity over the wild type, with one mutant, p97(A232E), having three times higher activity. Further mutagenesis of p97(A232E) shows that the increase in ATPase activity is mediated through D2 and requires both the N-domain and a flexible ND1 linker. A disulfide mutation that locks the N-domain to D1 in a coplanar position reversibly abrogates ATPase activity. A cryo-EM reconstruction of p97(A232E) suggests that the N-domains are flexible. Removal of the C-terminal region also reduces ATPase activity. Taken together, our data suggest that the conformation of the N-domain in relation to the D1-D2 hexamer is directly linked to ATP hydrolysis and that the C-terminal region is required for hexamer stability. This leads us to propose a model where the N-domain adopts either of two conformations: a flexible conformation compatible with ATP hydrolysis or a coplanar conformation that is inactive.

FIGURE 1.

IBMPFD-linked mutants in p97. A, graphic representation of the p97 crystal structure showing the distribution of the IBMPFD-linked mutations. For clarity, only the N-domain (blue), the D1 domain (green), and the connecting linker (yellow) are shown. The neighboring subunits in the hexamer are shown in gray. B, typical purification profile from size-exclusion chromatography of wild-type p97 (black), p97^R155C (blue), and p97^A232E (red). The relative molecular masses (M^r) of the peaks were estimated according to protein standard markers. C, negative-stain electron micrographs and class averages for wild-type p97, p97^R155C, and p97^A232E with representative top-view class averages (inset). Class averages are the sum of 50 top-view images aligned and classified together by multivariate statistical analysis. Scale bar = 500 Å on the micrographs and 50 Å on the class averages. D, ATPase activities of wild-type p97 and ten IBMPFD mutants. The histogram shows the rates of ATP hydrolysis averaged from at least three independent measurements. Error bars indicate the mean ± S.D. The activities were normalized to that of wild-type. An SDS-PAGE gel of the final purified p97 proteins from size-exclusion chromatography is shown in the lower panel. 10 μl of each protein at 3 μm was loaded onto a 12% polyacrylamide gel followed by Coomassie Brilliant Blue staining. E, ATPase activities of p97^A232E with D1 (E305Q) or D2 (E578Q) Walker B mutations. Note that only the Walker B mutation in D2 reduces the activity of p97^A232E.

FIGURE 2.

Modeling and cryo-EM structural analysis of the p97^A232E IBMPFD-linked mutant. A, electrostatic surface potential of p97 around A232. Hydrophobic areas are shown in white, with positive potential in red and negative potential in blue. B, a close-up view of A232 modeled with a glutamic acid side chain. Shown are top (C) and side (D) views of the cryo-EM reconstruction of p97^A232E at ∼23 Å. Overall dimensions are indicated. The density threshold was set to account for the molecular weight of p97^A232E. A model of D1-D2 derived from the p97 crystal structure has been fitted into the EM envelope. The reconstruction model shows two stacked rings and a cap of density at the top. The small bottom plug is likely to be an accumulation of noise around the symmetry axis, although it may potentially be the flexible C-terminal regions. The fitted D1 and D2 domains are colored in green and cyan, respectively. E, cryo-EM class average of the top and side views of p97^A232E. Scale bar = 50 Å.

FIGURE 3.

ATPase activity of p97^A232E without a flexible N-domain. A, ATPase activities of p97^A232E combined with an N-domain truncation or an N-D1 linker mutation. Measurements and presentation of results were done as for Fig. 1D. The D1 and D2 Walker B double-mutated p97ΔN was used as a negative control. B, alignment of the conserved Gly-Gly sequences in the N-D1 and D1-D2 linker regions among p97 homologs. The Gly-Gly sequences and Walker A motifs are highlighted.

FIGURE 4.

ATPase activity of the disulfide-linked p97^R159C/N387C mutant. A, size-exclusion chromatographic profiles of wild-type p97 (dotted line) and the p97^R159C/N387C cross-linked mutant (solid line). B, cryo-EM class average showing a side view of p97^R159C/N387C in the absence of DTT (left) and back projections of the p97 crystal structure filtered to 25 Å resolution, with (center) and without (right) the N-domain. Scale bars = 50 Å. C, ATPase activity of p97^R159C/N387C in the presence or absence of DTT. Measurements and representation of the histogram are the same as in Fig. 1D. Note that for p97^E305Q/E578Q (D1 and D2 Walker B double mutant) there is no difference in activity with and without DTT.

FIGURE 5.

ATPase activity of a C-terminal truncated p97. A, ATPase activities of p97ΔC and p97ΔC^A232E. Measurements and representation of the histogram are the same as in Fig. 1D. B, typical purification profiles from size-exclusion chromatography of the wild type (dotted line) and ΔC (solid line) as shown in Fig. 1B. C, limited proteolysis for wild-type p97 and p97ΔC in the presence or absence of ATPγS. Reactions were analyzed by SDS-PAGE. Shown are lane 1, no trypsin; lanes 2-6, incubation with trypsin for 0, 10, 20, 30, and 40 min, respectively. D, class averages of wild-type p97 and p97ΔC cryo-EM from the top and side views with respect to the 6-fold axis. Scale bar = 100 Å. Note the wider opened D1 ring of p97ΔC compared with that of the wild type.

FIGURE 6.

Structural model of N-domain flexibility. p97 can adopt two conformations in the ATPase cycle. In the “flexible” conformation, the D2 domains form a compact ring and p97 hydrolyzes ATP. When N-domains are coplanar with the D1 ring, p97 is unable to hydrolyze ATP. Only two subunits of the hexamer are shown in a side view, colored as in Figs. 1A and 2D.