Abstract
Human AAA+ protein p97 consists of an N-domain and two tandem ATPase domains D1 and D2, which are connected by the N-D1 and the D1-D2 linkers. Inclusion of the D1-D2 linker, a 22-amino acid peptide, at the end of p97 N-D1 truncate has been shown to activate ATP hydrolysis of its D1-domain, although the mechanism of activation remains unclear. Here, we identify the N-terminal half of this linker, highly conserved from human to fungi, is essential for the ATPase activation. By analyzing available crystal structures, we observed that the D1-D2 linker is capable of inducing asymmetry in subunit association into a p97 hexamer. This observation is reinforced by two new crystal structures, determined in the present work. The effect of D1-D2 linker on the ATPase activity of the D1-domain is correlated to the side-chain conformation of residue R359, a trans-acting arginine-finger residue essential for ATP hydrolysis of the D1-domain. The activation in D1-domain ATPase activity by breaking perfect six-fold symmetry implies functional importance of asymmetric association of p97 subunits, the extent of which can be determined quantitatively by the metric Asymmetric Index.
Highlights
In the structure of FLp97, the D1-domain ends at residue S459 and is followed by the D1-D2 linker (N460-G481), which is fully ordered and has been modeled as a random loop with no apparent secondary structure[3]
Reviewing all crystal structures of hexameric p97 available in the Protein Databank (PDB) determined in various lengths, nucleotide states and in the absence of adaptor proteins, we found it interesting that structures of p97 containing the D1-D2 linker were usually determined from crystals with lower symmetry (Table 1)
The phenomenon of asymmetry in structures of AAA+ proteins and its relation to function has been extensively documented, especially for those with asymmetry induced by binding of nucleotides
Summary
The N-terminal half of the D1-D2 linker is critical to the D1-domain activity. To shed more light on the mechanism of the D1-D2 linker in activating the ATPase activity of the D1-domain and identify the segment that is responsible, we generated a series of N-D1 truncates containing different lengths of the D1-D2 linker, and measured their ATPase activities. In the highest resolution structure containing the R155H mutation and with bound ATPγ S (PDB:4KO8 at 1.98 Å resolution), one out of two subunits in the asymmetric unit has the D1-D2 linker traced up to residue V469 (Fig. 3B). To test the above two possibilities, we crystallized wild-type ND1p97Lng with the D1-D2 linker (residues 1–481) in the presence of ADP This wild-type ND1p97Lng construct crystallized in the low symmetry space group P212121 with 12 subunits per asymmetric unit forming two hexamers (Tables 1 and 2), which is very different from the corresponding short form ND1p97Shrt that crystallized in the high symmetry space group P622 (PDB:1E32) (Table 1). The Asymmetric Index: a measurement for the asymmetric association of p97 subunits
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