title: Multifunctional roles of the conserved Arg residues in the second region of homology of p97/valosin-containing protein.
abstract: The 97-kDa molecular chaperone valosin-containing protein (VCP) belongs to a highly conserved AAA family and forms a hexameric structure that is essential for its biological functions. The AAA domain contains highly conserved motifs, the Walker A, Walker B, and the second region of homology (SRH). Although Walker A and B motifs mediate ATP binding and hydrolysis, respectively, the function of the SRH in VCP is not clear. We examined the significance of the SRH in VCP, especially the conserved Arg(359) and Arg(362) in the first AAA domain, D1 and Arg(635) and Arg(638) in the second AAA domain, D2. We show that Arg(359) and Arg(362) in D1 are critical for maintaining the hexameric structure and the ability to bind the polyubiquitin chains. Although the rest of the tested SRH mutants retain the hexameric structure, all of them exhibit severely reduced ATPase activity. Tryptophan fluorescence analysis showed that all of the tested mutants can bind to ATP or ADP. Thus, the reduced ATPase activity likely results from the hampered communications among protomers during hydrolysis. Moreover, when the ATPase-defective mutant R635A or R638A is mixed with the Walker A mutant of D2, the ATPase activity is partially restored, suggesting that Arg(635) and Arg(638) can stimulate the ATPase activity of the neighboring protomer. Interestingly, mutation of Arg(359) and Arg(362) uncouples the inhibitory effect of p47, a VCP co-factor, on the ATPase activity of VCP. Therefore, the Arg residues allow D1 to take on a specific conformation that is required for substrate binding and co-factor communications. Taken together, these results demonstrate that the conserved Arg residues in the SRH of both D1 and D2 play critical roles in communicating the conformational changes required for ATP hydrolysis, and SRH in D1 also contributes to substrate binding and co-factor communications.
authors.affiliation: Laboratory of Cancer Prevention, National Cancer Institute at Frederick, National Institutes of Health, Frederick, Maryland 21702, USA. qwang@ncifcrf.gov
authors.forename: Qing
authors.initials: Q
authors.lastname: Wang
authors.forename: Changcheng
authors.initials: C
authors.lastname: Song
authors.forename: Lauren
authors.initials: L
authors.lastname: Irizarry
authors.forename: Renming
authors.initials: R
authors.lastname: Dai
authors.forename: Xiaodong
authors.initials: X
authors.lastname: Zhang
authors.forename: Chou-Chi H
authors.initials: CC
authors.lastname: Li
mesh_terms: D000251:Adenosine Triphosphatases / Q000235:genetics / Q000378:metabolism; D000255:Adenosine Triphosphate / Q000378:metabolism; D000595:Amino Acid Sequence; D001120:Arginine / Q000737:chemistry / Q000235:genetics / Q000502:physiology*; D018797:Cell Cycle Proteins / Q000737:chemistry* / Q000235:genetics / Q000502:physiology; D017124:Conserved Sequence; D006868:Hydrolysis; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D025821:Polyubiquitin / Q000378:metabolism; D011487:Protein Conformation; D011993:Recombinant Fusion Proteins; D017385:Sequence Homology; D013050:Spectrometry, Fluorescence; D013329:Structure-Activity Relationship; D014364:Tryptophan / Q000737:chemistry; D000074405:Valosin Containing Protein
chemical_list: D018797:Cell Cycle Proteins; D011993:Recombinant Fusion Proteins; D025821:Polyubiquitin; D014364:Tryptophan; D000255:Adenosine Triphosphate; D001120:Arginine; D000251:Adenosine Triphosphatases; D000074405:Valosin Containing Protein