FIGURE 4.

Comparison of the non-covalent ubiquitin-binding surfaces of Ube2g1 and Ube2g1^Y102G/Y104G. A, ubiquitin binding surfaces of Ube2g1 as determined by CSP experiments in the presence of 0.5 mm ubiquitin. The magenta, red, orange, and yellow colors, respectively, indicate high to low magnitude of CSP. The peaks of Phe-25, Ala-27, and Ser-126 disappeared upon the addition of 0.5 mm ubiquitin and are in green. Residues in black represent prolines, which are not visible in the HSQC spectrum due to the absence of an amide proton. The original peak intensity of Leru-112 was too weak to determine the CSP and is, therefore, also in black. The ubiquitin binding surface of Ube2g1 can be separated into three parts, namely the bottom, L-side, and loop. The non-covalent ubiquitin binding affinities (K_d values) were measured by titrations with ubiquitin to be 1.25 ± 0.39, 1.32 ± 0.40, and 1.46 ± 0.36 mm for the bottom, L-side, and loop regions, respectively. B, the CSP data for [¹⁵N]Ube2g1 and [¹⁵N]Ube2g1^Y102G/Y104G in the presence of 0.5 mm ubiquitin are represented as a histogram. The striped bars represent residues for which the resonance peaks disappeared in the presence of 0.5 mm ubiquitin. The negative black and gray bars represent, respectively, Pro residues or residues where peak intensities were too weak to be detected in the CSP experiment. Note that Ube2g1^Y102G/Y104G lost ubiquitin interactions only in the acidic loop region.