Specificity of the E1-E2-E3 Enzymatic Cascade for Ubiquitin C-Terminal Sequences Identified by Phage Display

Abstract

Ubiquitin (UB) is a protein modifier that regulates many essential cellular processes. To initiate protein modification by UB, the E1 enzyme activates the C-terminal carboxylate of UB to launch its transfer through the E1-E2-E3 cascade onto target proteins. In this study, we used phage display to profile the specificity of the two human E1 enzymes, Ube1 and Uba6, towards the C-terminal sequence of UB ending with 71LRLRGG76. Phage selection revealed that while Arg72 of UB is absolutely required for E1 recognition, UB residues at positions 71, 73 and 74 can be replaced with bulky aromatic side chains, and Gly75 of UB can be changed to Ser, Asp and Asn for efficient E1 activation. We have thus found that the E1 enzymes have substantial promiscuity regarding the UB C-terminal sequence. The UB variants from phage selection can also be transferred from E1 to E2 enzymes, however, they are blocked from further transfer to the E3 enzymes. This suggests that the C-terminal sequence of UB is important for its discharge from E2 and subsequent transfer to E3. In addition, we observed that the Leu73Phe and Leu73Tyr single mutants of UB are resistant to cleavage by deubiquitinating enzymes (DUBs), although they can be assembled by the E1-E2-E3 cascade into poly-UB chains, thus indicating differences in UB C-terminal specificities between the E1 and DUBs. Consequently these UB mutants may provide stability to UB polymers attached to cellular proteins and facilitate the elucidation of the biological signals encoded in the UB chains.

Systematic Exploration of Ubiquitin Sequence, E1 Activation Efficiency, and Experimental Fitness in Yeast

“…We did not observe reaction of G75D ubiquitin with E1 even at concentrations 100-fold greater than those where we observed reaction with wild type, or 10-fold greater than for the R72S variant. The G75D ubiquitin variant was recently recovered in a phage display selection for E1 reactivity 47 , which may be due to the use of non-covalent and unstable 48 Fos-Jun mediated association between ubiquitin and phage particles, or other distinctions between the experimental setups. Our observations with purified proteins show that purified G75D ubiquitin was severely defective for E1 reactivity.…”

Profiling the Cross Reactivity of Ubiquitin with the Nedd8 Activating Enzyme by Phage Display

“…In case of all three residues (71, 73 and 74), there is significantly more open space around their side chains compared to the residue at position 72. This might explain the enrichment of aromatic residues at positions 71, 73 and 74 when the UB library was selected with NAE in this study and selected with UAE in an earlier study [27].…”

“…We next carried out phage selection of a UB library that was constructed in a previous work with randomized residues replacing 71 LRLRG 75 at the UB C-terminus [27]. Selection of the phage library was based on the formation of thioester conjugates between phage displayed UB variants and NAE immobilized on the streptavidin plate (Figure 1b).…”

“…Since we previously used UAE to select for reactive UB variants from the same library [27], we can now compare the profiles of UB C-terminal sequences that are recognized by NAE and UAE, respectively. The sequence alignment of UB variants from UAE selection showed that all selected UB clones retained Arg72 in the wt UB clearly indicating that the positively charged Arg side chain at this position is essential for UAE recognition [27]. In contrast UB variants from NAE selection have Arg72 of wt UB replaced by hydrophobic side chains of variable sizes such as Ala, Val, Leu, Ile, Phe, Tyr, and Met or, more infrequently, polar side chains Ser or Gln (Figure 3).…”

“…Still Gly75 can be replaced by Ser for NAE recognition, and by Ser, Asp, Asn and Gln for UAE recognition [27].…”