Figure 5: Characterization of the ENTH–Ub binding interface.

Structure-based mutants at the predicted ENTH–Ub interface were constructed and characterized. (a) Growth phenotypes of the zebrafish ENTH and Ub mutants in the selection system. Bacteria expressing a complete (pND–Ub, E1, yeast Ubc4 and pCD–ENTH variants) or incomplete apparatus (ΔE1,ΔE2 or ΔUb) were seeded and visualized as in Figure 1. (b) Growth curves of Ub wild type (WT) and mutants derived from the timelapse scanning of the spots (density was analyzed by Fiji). (c) As shown in b, but for ENTH mutants. (d) As shown in b and c, but for reciprocal mutations. (e) Growth phenotypes of the yeast ENTH–Ub interface mutants in the selection system. Bacterial spots were seeded and visualized as in Figure 1. (f) As shown in b, but for yeast ENTH and Ub mutants. (g) The ubiquitylation yield for wild type and indicated mutants of the yeast His₆–MBP–ENTH were evaluated. The apo and ubiquitylated proteins were purified on amylose beads as described¹⁷, resolved on SDS–PAGE and detected by western blot with anti-His-tag antibody. (h) Quantification of the ubiquitylation yields shown in g. The ubiquitylated/apo ratio is presented as a percentage of the wild-type ratio. Values were averaged from four independent experiments, and s.d. error bars are presented. (i) Surface plasmon resonance (SPR) analysis of the yeast ENTH–Ub binding affinity of wild-type and mutant proteins. Fitting to binding curves was carried out with a single-site-binding model using the OriginLab software. Standard errors derived from three independent measurements are indicated.