Human UBC9 is a member of the E2 (ubiquitin conjugation enzyme) family of proteins. Instead of conjugating to ubiquitin, it conjugates with a ubiquitin homologue UBL1 (also known as SUMO-1, GMP1, SMTP3, PIC1, and sentrin). UBC9 has been shown to be involved in cell cycle regulation, DNA repair, and p53-dependent processes. The binding interfaces of the UBC9 and UBL1 complex have been determined by chemical shift perturbation using nuclear magnetic resonance spectroscopy. The binding site of UBL1 resides on the ubiquitin domain, and the binding site of UBC9 is located on a structurally conserved region of E2 . Because the UBC9-UBL1 system shares many similarities with the ubiquitin system in structures and in conjugation with each other and with target proteins, the observed binding interfaces may be conserved in E2-ubiquitin interactions in general.The ubiquitin pathway is crucial in cellular regulation (1-3). It is now clear that this pathway regulates the life span of many important proteins, including some cyclins, cyclin-dependent kinase inhibitors, histones, oncoproteins, and tumor suppressors. Therefore, this pathway regulates many cellular processes, including cell cycle progression and apoptosis, transcription regulation, and antigen presentation (4 -6). Ubiquitin is a protein with 76 amino acids. Its C-terminal Gly residue is involved in covalent conjugation to a Lys residue of other proteins. The C-terminal Gly residue of one ubiquitin molecule can also conjugate to Lys 48 of another ubiquitin molecule in multiubiquitination. In the ubiquitination pathway, the ubiquitin activation enzyme E1 1 activates ubiquitin by hydrolyzing ATP to form a high energy bond with ubiquitin. Ubiquitin is then transferred to a ubiquitin conjugation enzyme (UBC) also known as E2. In this step, the C-terminal Gly is conjugated to the SH group of the active-site Cys residue of E2. Then E2 interacts with substrate proteins to transfer ubiquitin to the substrate proteins. In some cases, this process requires the participation of ubiquitin-protein ligase (E3). Proteins with high sequence homology to ubiquitin and to the E1 and E2 enzymes have been found.Human UBC9, composed of 158 amino acid residues, is a member of the E2 protein family. Among the conserved residues in the E2 family of proteins, the fragment containing the ubiquitin-accepting Cys residue is highly conserved. UBC9 conjugates with a ubiquitin homologue, UBL1, instead of ubiquitin. The interaction between UBC9 and UBL1 is specific, because UBC9 does not interact well with ubiquitin, and UBL1 does not interact with UBC2 in a yeast two-hybrid system (7-9). UBC9 in yeast, mouse, and human have been identified (10, 11, 7). Many proteins interact with UBC9. The human UBC9 has been shown to interact with several important proteins, such as DNA repair proteins RAD51 and RAD52, p53, c-JUN, glucocorticoid receptor, the negative regulatory domain of the Wilms' tumor gene product, and human papillomavirus type 16 E1 replication protein (12)(13)(14). Seufert et al. (10) sho...