Direct characterization of E2-dependent target specificity and processivity using an artificial p27-linker-E2 ubiquitination system

Abstract

Little attention has been paid to the specificity between E2 and the target protein during ubiquitination, although RING-E3 induces a potential intra-molecular reaction by mediating the direct transfer of ubiquitin from E2 to the target protein. We have constructed artificial E2 fusion proteins in which a target protein (p27) is tethered to one of six E2s via a flexible linker. Interestingly, only three E2s (UbcH5b, hHR6b, and Cdc34) are able to ubiquitinate p27 via an intra-molecular reaction in this system. Although the first ubiquitination of p27 (p27-Ub) by Cdc34 is less efficient than that of UbcH5b and hHR6b, the additional ubiquitin attachment to p27-Ub by Cdc34 is highly efficient. The E2 core of Cdc34 provides specificity to p27, and the residues 184-196 are required for possessive ubiquitination by Cdc34. We demonstrate direct E2 specificity for p27 and also show that differential ubiquitin linkages can be dependent on E2 alone. [BMB reports 2008; 41(12): 852-857]

Differential Ubiquitin Binding by the Acidic Loops of Ube2g1 and Ube2r1 Enzymes Distinguishes Their Lys-48-ubiquitylation Activities

“…12, see gel band indicated as Di*); this suggests that the acidic loop also may play an important role in proper alignment of an acceptor ubiquitin. The production of non-Lys-48-linked diubiquitin was reported for Ube2d2 and Ube2b in the absence of E3 (8).…”

“…Target proteins were further purified by gel permeation chromatography using Superdex-75 (GE Healthcare); where applicable, subsequent passage through a GSTrap column was used to remove the cleaved GST. Murine E1 protein was purified following a previously reported method (8), and the concentration of the E1 stock solution (ϳ2.0 mg/ml) was estimated based on band intensity observed after sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and staining with Coomassie Blue.…”

“…Ube2g1 (12), although Ube2d2 lacks an acidic loop and also cannot synthesize Lys-48 polyubiquitin chains efficiently in the absence of an E3 (8). We now have compared the ubiquitininteracting surfaces of Ube2g1 and Ube2d2 in detail.…”

“…Ube2g1 consists of only an E2-core domain, but Ube2r1 has an additional C-terminal extension (residues 184 -236). Work from our group previously showed that, unlike full-length Ube2r1, the Ube2r1 core (residues 1-183; Ube2r1C) has a decreased capability to synthesize Lys-48 polyubiquitin chains efficiently in the absence of an E3 (8). Ube2d2, which does not have an acidic loop, also is unable to synthesize Lys-48 polyubiquitin in the absence of an E3 ligase (8) despite having ubiquitin binding affinity almost identical to that of Ube2g1 as assessed by NMR CSP experiments using [ 15 N]UB (12).…”

“…An in vitro E3-independent ubiquitylation assay that used p27-E2 fusion proteins had identified Ube2d2 (UbcH5b) and Ube2b (hHR6b) as the most efficient mediators for the ubiquitylation of p27, but those E2s were found to be less effective for subsequent Lys-48-ubiquitylation in comparison to Ube2r1 (human Cdc34) (8). Recent reports showed that the sequential engagement of two different E2 enzymes may be important for efficient polyubiquitylation of target proteins in an E3-dependent system.…”