Figure 2: Identification and characterization of NleG6-3 as E3 ligase.

Applying the genetic selection system for identification and characterization of E3 ligases. Bacterial spots were seeded and visualized as in Figure 1. (a) Bacteria that coexpress cDHFR fusion to the E3 ligase Rsp5 (representative of the HECT family) with its cognate ubiquitylation apparatus including Ubc4 are labeled 'complete'. Deletions of E1, E2 or Ub are indicated. (b) Bacteria that coexpress cDHFR fusion to the E3 ligase SIAH2 (representative of the RING family) with its cognate ubiquitylation apparatus, including UBCH5A, are labeled 'complete'. Deletions of E1, E2, Ub or substitution of the cognate E2 with Cdc34 (a noncognate E2) are indicated. The right panel shows the effect of the small-molecule inhibitor, menadione, on SIAH2-dependent bacterial growth. (c) Sequence alignment derived from PSI-BLAST search using human CHIP as probe against the EHEC proteome. (d) Yeast E2s scan for the E3 ligase ECs3488 (NleG6-3). Self ubiquitylation of the potential E3 ligase was examined against the indicated E2s. (e) Self ubiquitylation of NleG6-3 by human UBCH5B and UBCH5C. (f) Homology-based model of NleG6-3 (orange) superimposed on the structure of EHEC NleG2-3 (blue) and Human CHIP–UBCH5B complex (gray and magenta, respectively), showing a zoomed-in view of the predicted interface. (g) Employment of the selection system for mutational analysis of the predicted E2–E3 interface. Growth of bacterial spots of the indicated mutants on the predicted E3-ligase surface coexpressed with E1 and UBCH5B are shown.