FIGURE 2.

Parkin Ubld interacts with UIM I in S5a. Selected regions of the 600 MHz ¹H-¹⁵N HSQC spectra showing the interaction of the parkin Ubld with ¹⁵N-labeled (A) S5a-(196–309) or (B) S5a-(196–309)^UIMII-5A. In both figures the S5a-(196–309) (or S5a-(196–309)^UIMII-5A) is shown in the absence of parkin Ubld (black contours) and presence of 2 eq of parkin Ubld (red contours). The spectra were collected using 500 μm S5a protein in 10 mm KH₂PO₄, 1 mm EDTA, 1 mm dithiothreitol, 150 mm NaCl, pH 7, at 25 °C. The spectrum of S5a-(196–309) was assigned according to triple resonance experiments and published work (44). The spectrum of S5a-(196–309)^UIMII-5A was assigned by triple resonance NMR methods. Spectra are presented at different contour levels to account for line broadening in the S5a proteins upon Ubld addition. Binding curves based on NMR titration data are shown in C and D. In C the S5a-(196–309) titration data is plotted and fit using peaks near UIM I (Ser²¹¹ and Ala²¹²) and UIM II (Met²⁹¹ and Met²⁹³) In D, the S5a-(196–309)^UIMII-5A titration data are plotted using residues from UIM I only (Ser²¹¹ and Ala²¹²). The sequence of the S5a UIM regions is shown in E with the portion of the linker region between Arg²³⁰ and Lys²⁶² omitted for clarity.