Hereditary inclusion body myopathy-linked p97/VCP mutations in the NH2 domain and the D1 ring modulate p97/VCP ATPase activity and D2 ring conformation.
Hereditary inclusion body myopathy associated with early-onset Paget disease of bone and frontotemporal dementia (hIBMPFTD) is a degenerative disorder caused by single substitutions in highly conserved residues of p97/VCP. All mutations identified thus far cluster within the NH(2) domain or the D1 ring, which are both required for communicating conformational changes to adaptor protein complexes. In this study, biochemical approaches were used to identify the consequences of the mutations R155P and A232E on p97/VCP structure. Assessment of p97/VCP oligomerization revealed that p97(R155P) and p97(A232E) formed hexameric ring-shaped structures of approximately 600 kDa. p97(R155P) and p97(A232E) exhibited an approximately 3-fold increase in ATPase activity compared to wild-type p97 (p97(WT)) and displayed increased sensitivity to heat-induced upregulation of ATPase activity. Protein fluorescence analysis provided evidence for conformational differences in the D2 rings of both hIBMPFTD mutants. Furthermore, both mutations increased the proteolytic susceptibility of the D2 ring. The solution structures of all p97/VCP proteins revealed a didispersed distribution of a predominant hexameric population and a minor population of large-diameter complexes. ATP binding significantly increased the abundance of large-diameter complexes for p97(R155P) and p97(A232E), but not p97(WT) or the ATP-binding mutant p97(K524A). Therefore, we propose that hIBMPFTD p97/VCP mutants p97(R155P) and p97(A232E) possess structural defects that may compromise the mechanism of p97/VCP activity within large multiprotein complexes.
Halawani
Dalia
D
The Nicholas Conor Institute for Pediatric Cancer Research, 9710 Scranton Road, Suite 170, San Diego, CA 92121, USA.
LeBlanc
Andréa C
AC
Rouiller
Isabelle
I
Michnick
Stephen W
SW
Servant
Marc J
MJ
Latterich
Martin
M
D000251:Adenosine Triphosphatases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D000255:Adenosine Triphosphate / Q000378:metabolism; D018797:Cell Cycle Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D006801:Humans; D046912:Multiprotein Complexes / Q000737:chemistry / Q000378:metabolism; D009154:Mutation; D018979:Myositis, Inclusion Body / Q000235:genetics / Q000378:metabolism; D010316:Particle Size; D010641:Phenotype; D011487:Protein Conformation; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D000074405:Valosin Containing Protein
D018797:Cell Cycle Proteins; D046912:Multiprotein Complexes; D011993:Recombinant Fusion Proteins; D000255:Adenosine Triphosphate; D000251:Adenosine Triphosphatases; C000614178:VCP protein, human; D000074405:Valosin Containing Protein