FIGURE 4.

The Ile-44 hydrophobic patch of ubiquitin is required for binding. A, the indicated His₆-tagged ubiquitin mutants were immobilized on beads and incubated with a bacterial lysate from cells expressing a wild-type GST-tagged N-lobe. Bound proteins and ubiquitin mutants eluted from the beads were detected by Coomassie staining. B, surface representation of ubiquitin based on its three-dimensional structure (Protein Data Bank accession code 1UBQ). Results from the alanine mutagenesis are summarized as follows: red, mutation abolished binding; magenta, mutation reduced binding; dark gray, mutation had no effect. The position of Lys-63 is shown for reference. No mutations were made on the back face of ubiquitin (not shown).