We find that Ubc12’s unique N-terminal extension tethers Ubc12 selectively to APPBP1-UBA3 via a novel type of E1–E2 interaction. First, deletion of Ubc12’s N-terminal extension reduces the Km, but not kcat, for APPBP1-UBA3-catalyzed Ubc12~NEDD8 thioester formation. Second, a peptide corresponding to Ubc12’s N-terminal 26 residues inhibits NEDD8~Ubc12 thioester formation. This effect is specific to the NEDD8 pathway, because the peptide does not affect thioester formation between ubiquitin or Sumo and their E2s (). Third, deletion mutations reveal a minimum length requirement for the linker between Ubc12’s docking peptide and E2 core domain. These results suggest that for the NEDD8 pathway, the E1–E2 interaction is bipartite: both Ubc12’s docking peptide and catalytic core domain must bind the E1 simultaneously for optimal transfer of NEDD8 from E1 to E2.
The crystal structure of the APPBP1-UBA3-Ubc12N26 peptide complex reveals that Ubc12’s peptide-like extension interacts with a docking groove generated from unique loop sequences in UBA3. Even though the docking peptide and docking groove sequences are unique to Ubc12 and UBA3 family members, respectively, the adenylation domain portion of UBA3 that contains the docking groove corresponds to the most conserved domain in UBL activating enzymes13,32
. Thus, the structure reveals how unique protein-protein interactions, specific for a particular UBL’s pathway, can be generated from a common structural scaffold such as an E1 adenylation domain.
Many post-translational modifications are directed to their targets via multi-enzyme cascades. In addition to UBL modification cascades, some of the best-studied examples of multi-enzyme post-translational modification cascades are serine/threonine phosphorylation pathways, such as MAPK and cyclin-dependent kinase (cdk) cascades. Several characteristics of the interaction between NEDD8’s E1 and the peptide from NEDD8’s E2 are reminiscent of interactions in these pathways. MAP kinases and cdks bind their targets through docking peptide-docking groove interactions33,34
. Similar to APPBP1-UBA3’s interactions with Ubc12, the interactions between MAPKs and cdks with the downstream enzymes in their cascades are often bipartite, with both a docking peptide binding distal from the active site and the target’s phosphoacceptor sequence binding at the kinase active site. A primary function of the docking peptide interaction with these kinases is to reduce the Km
of the target35
. Thus, the APPBP1-UBA3-Ubc12N26 interaction reveals that common design principles underlie these very divergent multi-enzyme post-translational modification cascades.
The E1–E2[N-terminal peptide] interaction found between APPBP1-UBA3 and Ubc12 is likely to be unique to the NEDD8 pathway, because many E2s for other UBLs lack N-terminal extensions. We wonder why the NEDD8 cascade requires additional E1–E2 interactions. Clues to a possible function for this interaction come from the paradigm of docking groove – docking peptide interactions established by serine/threonine phosphorylation cascades. In MAP kinase or cdk cascades, the kinase docking groove is multifunctional, recruiting not only targets, but also regulatory proteins such as other kinases, phosphatases, and inhibitors, which all contain similar docking peptide sequences33,34
. For example, part of the function of some cdk inhibitors comes from their ability to displace peptide-like docking sequences in substrates33
. Interestingly, a UBA3 binding protein, But1, has recently been identified in fission yeast36
. But1’s biological function is consistent with an inhibitory role in the NEDD8 pathway36
. The But1 sequence contains many hydrophobic-hydrophobic-X-hydrophobic sequences similar to the Leu4-Phe5-Ser6-Leu7 sequence that we find anchors Ubc12 in UBA3’s docking groove. Future studies will reveal whether But1 or other proteins bind to UBA3 via the Ubc12 docking groove to inhibit NEDD8 conjugation.
Another possible function for the additional APPBP1-UBA3-Ubc12N26 interaction comes from the remarkable specificity of the NEDD8 pathway2–4
. NEDD8 modification of cullins is one of many regulatory mechanisms controlling ubiquitin conjugation by SCF and related E3s. Consistent with its important regulatory function, NEDD8 modification of cullins is controlled not only by the conjugation pathway, but also by deconjugation by the COP9 signalosome37
, underscoring the importance of precision in this pathway. The additional E1–E2 interaction may serve to ensure the selectivity of the NEDD8 conjugation cascade.
Like Ubc12, many other E2s also have their own distinct extensions at their N- and C-termini, beyond the conserved E2 core domain. The molecular functions of only a handful of these extensions are known38–41
, and to date, the structural basis for protein-protein interactions mediated by these extensions remain elusive. One of the best understood extensions is in the E2 Ubc2p, which interacts with the E3, Ubr1p. While the Ubc2p-Ubr1p interaction is likely to involve interactions common to E2-E3 complexes because it requires Ubr1p’s RING domain, Ubc2p’s C-terminal extension is also known to stabilize the interaction with Ubr1p42
. Therefore, it is likely that the extensions on other E2s function in a manner analogous to Ubc12’s, strengthening interactions between enzymes in other UBL modification cascades.
The importance of Ubc12’s N-terminal extension is further underscored by the detrimental effect of the Ubc12ΔN deletion in our cell proliferation assay. The best-characterized targets of Ubc12 are cullins2–4,12,9,5,6,10,11,7,8,43
, although we do not yet know which targets are important in our assay. Cul1, Cul2, Cul3 and Cul4a-containing ubiquitin ligases are known to promote the degradation of key regulators of cell proliferation, including cyclins, cyclin-dependent kinase inhibitors, proteins involved in DNA replication, proteins involved in mitotic spindle assembly, transcription factors, and proteins involved in signal transduction (reviewed in 43
). It will be of interest to determine which targets of Ubc12-mediated NEDD8 conjugation are involved in the CSF-1-dependent proliferation. Our finding that Ubc12ΔN is defective in this cell proliferation assay raises the possibility that APPBP1-UBA3 and Ubc12 may serve as good targets for antimitogenic agents.