Individual
UIM regions in ataxin-3 bind similarly to the parkin
Ubl domain. Regions of 1H–15N HSQC spectra
of the 15N-labeled parkin Ubl domain (100 μM) showing
selected residues that exhibit changes in chemical shifts upon addition
of (A) UIM_1, (B) UIM_2, and (C) UIM_3. The change in chemical shift
as a function of increasing ataxin-3 protein concentration is shown
for residues F13, K48, V67, and Q64. The middle panels show the binding
curves for (A) UIM_1, (B) UIM_2, and (C) UIM_3 with the parkin Ubl
domain derived from NMR data. The data were globally fit for 1:1 binding
using eq 2 to determine the apparent dissociation
constants; KD,UIM_I = 840 ± 189 μM, KD,UIM_2 = 502 ± 41 μM, and KD,UIM_3 = 921 ± 165 μM. In each case,
fits were completed using residues K48 (▲), E49 (⬣),
H68 (○), Q64 (◆), L61 (□), and V43 (∗).
The right panel depicts the corresponding surface representations
of the parkin Ubl domain (Protein Data Bank entry 1IYF) showing residues
affected by binding of UIM_1 (purple), UIM_2 (cyan), and UIM_3 (orange).
The surface representations were created in PyMOL (PyMOL Molecular
Graphics System, version 1.5.0.4, Schrödinger, LLC).