The Human Cdc34 Carboxyl Terminus Contains a Non-covalent Ubiquitin Binding Activity That Contributes to SCF-dependent Ubiquitination

Abstract

Cdc34 is an E2 ubiquitin-conjugating enzyme that functions in conjunction with SCF (Skp1⅐Cullin 1⅐F-box) E3 ubiquitin ligase to catalyze covalent attachment of polyubiquitin chains to a target protein.Here we identified direct interactions between the human Cdc34 C terminus and ubiquitin using NMR chemical shift perturbation assays. The ubiquitin binding activity was mapped to two separate Cdc34 C-terminal motifs (UBS1 and UBS2) that comprise residues 206-215 and 216-225, respectively. UBS1 and UBS2 bind to ubiquitin in the proximity of ubiquitin Lys 48 and C-terminal tail, both of which are key sites for conjugation. When bound to ubiquitin in one orientation, the Cdc34 UBS1 aromatic residues (Phe Ubiquitination is a complex protein modification reaction that produces a covalent isopeptide bond linking the ubiquitin Gly 76 carboxyl end to the ⑀-amino group of a lysine residue within a substrate protein (1). One of the hallmarks of this modification is that substrates are frequently attached with polyubiquitin chains, formed by isopeptide linkage joining the ubiquitin Gly 76 carboxyl end and one of its seven internal lysine residues. Three enzymatic activities typically participate in the ubiquitination reaction, including a ubiquitin-activating enzyme (E1) that forms an E1-Sϳubiquitin thiol ester complex in an ATP hydrolysis-dependent manner, a ubiquitin-conjugating enzyme (E2) capable of tethering a ubiquitin in thiol ester form that is transferred from E1, and a ubiquitin-ligase (E3). RING finger domain-containing E3s orchestrate the transfer of ubiquitin to a substrate by recruiting both the target protein and E2-Sϳubiquitin thiol ester and by helping position the attacking substrate lysine and the E2-bound donor ubiquitin optimally for conjugation (2). SCF (Skp1⅐Cullin⅐F-box) is a four-subunit RING E3 ligase complex with CUL1 (cullin 1) as scaffold that at its N terminus tethers the Skp1⅐F-box protein complex to target a substrate and utilizes its C terminus to dock the ROC1/Rbx1/Hrt1 RING finger subunit for recruiting an E2 ubiquitin-conjugating enzyme (3-9). SCF-mediated polyubiquitination is activated by conjugation of Nedd8, a ubiquitin-like protein, to human CUL1 at lysine 720 (10, 11), most likely due to Nedd8-imposed conformational changes in CUL1 (12), thereby relieving autoinhibitory interactions between the extreme C terminus of CUL1 and ROC1 (13).Accumulating evidence demonstrates a role for the Cdc34 E2 in SCF-dependent ubiquitination. Yeast Cdc34 (Ubc3) is essential for viability and is required for SCF Cdc4 -dependent degradation of the cyclin-dependent kinase inhibitor Sic1 (see Refs. 3 and 4 and references therein). This regulation has been recapitulated by in vitro experiments that reconstitute the SCF Cdcc4 -dependent and Cdc34-catalyzed ubiquitination of Sic1 and the degradation of the modified substrate by the 26 S proteasome (3,4,14). Human Cdc34 (hCdc34) 5 is highly conserved as it is

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

“…4, A (12). The acidic loop in Ube2g1 is similar to the previously characterized weak ubiquitin-binding site 1 (UBS1) in the C-terminal tail of Ube2r1 (DLFYDDYYED) (26), which also consists of hydrophobic and acidic residues (Fig. 1).…”

“…Acidic loop or tail motifs are known to be important for the ubiquitylation activities of E2 or E3 enzymes; examples are the acidic loops in Ube2g1 and Ube2g2 (36), the acidic tail (residues 888 -900) in human Nedd4 (HECT-E3) (37), and both acidic loop and acidic tail in Ube2r1 (10,26,38). These motifs possess both acidic and bulky hydrophobic residues and usually are near the active-site Cys residue.…”

“…The sequence alignment was performed through structural matching of the E2 enzymes using the Chimera program (24). The previously identified non-covalent ubiquitin-binding sites of the Ube2r1 C-terminal tail (26) are marked with a gray highlight, and the acidic loop region is indicated by a yellow highlight. The asterisk represents the active-site Cys residues of the E2 enzymes.…”

“…It has been reported that the C-terminal extension of Ube2r1 includes two weak ubiquitin binding motifs (UBS1, residues 205-214, and UBS2, residues 216 -225) and an additional possibility of a third, very weak ubiquitin-binding site (residues 187-196) (26). However, Ube2r1C alone did not detectably bind to [ 15 N]UB (data not shown); the low background with Ube2r1C made identification of ubiquitin binding differences between Ube2r1C and Ube2r1C YGY straightforward.…”

A SnapShot of Ubiquitin Chain Elongation

“…Upon thiolester formation of Cdc34, the proximity of the SCF Rbx1/ROC1 subunit favors an interaction with the Cdc34ϳUb conjugate (22), which likely promotes Ub transfer to a substrate. In this context, Cdc34 may utilize both the core domain (22) and C terminus (31,32) for interactions with Ub. A recent study has provided structural evidence suggesting that the RING/E2/Ub interactions position the Ub carboxyl tail in the E2 active site groove for catalysis (33).…”