The N-terminal Region of the Ubiquitin Regulatory X (UBX) Domain-containing Protein 1 (UBXD1) Modulates Interdomain Communication within the Valosin-containing Protein p97

Abstract

Background: p97 cooperates with cofactors to control various aspects of cellular homeostasis. Mutations at the interdomain interface cause a multisystem degenerative disorder. Results: We identified three binding epitopes on p97 for the N-terminal domain of cofactor UBXD1 (UBXD1-N), including disease-associated residues. Binding reduced p97 ATPase activity. Conclusion: UBXD1-N modulates interdomain communication and activity of p97. Significance: The polyvalent binding mode defines a new subset of p97 cofactors.

A Dynamic molecular basis for malfunction in disease mutants of p97/VCP

“…Larger shifts are observed for V68 and I175 that report on VIM binding, from which a $true0K_d$ value of 22 ± 2 µM for the VIM-NTD interaction is calculated (see Materials and methods Figure 7—figure supplement 3). This value is in good agreement with affinities measured for the binding of full-length wt p97 to full length UBXD1, 3.5 µM (Hänzelmann and Schindelin, 2011), and for the binding of the isolated NTD with UBXD1-N, 9 µM (Trusch et al, 2015), especially considering that the latter pair of studies were carried out at 25°C in comparison to 50°C for the NMR results reported here. Studies of R155H and N387H mutants of ND1Lp97-ADP that mildly perturb the up/down equilibrium (Figure 7C, Figure 7—figure supplement 2D) show that two-pronged binding involving VIM and H1/H2 occurs partially but that a complete down conformation is not obtained with a 3-fold excess of adaptor over p97 protomer (compare dashed and solid arrows that correspond to movements for peaks from wt and mutant p97, respectively).…”

“…For wt, moderate (R155H and N387H) and severe (R95G, R155P) mutants, $true0K_1$ << 1, $true0K_1$ ~ 0.15, $true0K_1$ ~ 1, respectively (Figure 6). A value of $true0K_2$ ~ 200 µM has been measured, as described above, and although a value for K 3 is not available from our studies (too weak) it has been measured to be approximately γ=25 fold larger than $true0K_2$ in studies of an isolated NTD (Trusch et al, 2015). We have carried out simulations assuming $true0K_5$=0.1 (wt), $true0K_5$=0.5 (R155H, N387H) and $true0K_5$=10 (R95G, R155P) ( i.e ., locking of the NTD becomes progressively more difficult with severity of mutation), with $true0K_3$ and $true0K_4$ obtained from $true0{\mathrm{K}}_2={\mathrm{K}}_3/\gamma$ and $true0{\mathrm{K}}_4={\mathrm{K}}_5/\gamma$, respectively, assuming γ=25 for wt and γ increasing for the mutants in proportion to $true0K_5$(mutant)/$true0K_5$(wt) so that $true0K_2$ and $true0K_4$ are fixed (microscopic binding of VIM unperturbed by mutation).…”

“…Importantly, the UBXD1-p97 complex is specifically disrupted in disease-associated mutations and function is impaired (Ritz et al, 2011) but the molecular details underlying this disruption are not understood. An elegant biophysical study of the isolated NTD with the N-terminal portion of UBXD1 (UBXD1-N, residues 1–133, Figure 7A) (Trusch et al, 2015) shows an interaction involving VIM (residues 52–63) that is localized to the canonical VIM/UBX binding site on the NTD (Stapf et al, 2011). Moreover, the authors suggest that the N-terminus of UBXD1 may engage the NTD-D1 interface and NTD-D1 linker, where disease mutations occur, and hypothesize that this secondary interaction may favor a more compact conformation of p97.…”

“…The truncations used for this study were p97 ND1L 1–480 (Song et al, 2003) and p97 NTD 1–213 (Isaacson et al, 2007). DNA encoding adaptors, including full-length p47, p47 UBX (residues 282–371 [Yuan et al, 2001]) and UBXD1-N (residues 1–133 [Trusch et al, 2015]) from Mus musculus were also obtained from GenScript, with affinity tags, TEV cleavage sites and sub-cloning as for p97.…”

“…A previous study has established that UBXD1-N has two p97 binding regions (Trusch et al, 2015). These include a well-characterized VIM domain that forms a single ~10 residue helix upon binding to a groove between NTD sub-domains (Stapf et al, 2011; Hänzelmann and Schindelin, 2011) and an H1/H2 helical domain that has been shown through biochemical studies to fix the NTDs of wt p97-ADP in the down position (referred in our work as the locked, down position).…”