Interaction of the tail with the catalytic region of a class II E2 conjugating enzyme

Ubiquitination plays an important role in many biological processes, including DNA repair, cell cycle regulation, and protein degradation. In the latter pathway the ubiquitin-conjugating enzymes or E2 enzymes are important proteins forming a key E2-ubiquitin thiolester prior to substrate labelling. While the structure of the 150-residue catalytic domain has been well characterized, a subset of E2 enzymes (class II) carry a variable length C-terminal `tail' where structural detail is not available. The presence of this C-terminal extension plays an important role in target recognition, ubiquitin chain assembly and oligomerization. In this work NMR spectroscopy was used to determine the secondary structure of the 215-residue yeast E2 protein Ubc1 and the interactions of its C-terminus with the catalytic domain. The C-terminal tail of Ubc1 was found to contain three -helices between residues D169-S176, K183-L193 and N203-L213 providing the first evidence for a well-defined secondary structure in this region. Chemical shift mapping indicated that residues in the L2 loop of the catalytic domain were most affected indicating the C-terminus of Ubc1 likely interacts with this region. This site of interaction is distinct from that observed in the E2-ubiquitin thiolester and may act to protect the catalytic C88 residue and direct the interaction of ubiquitin in the thiolester intermediate.