Molecular and structural insight into lysine selection on substrate and ubiquitin lysine 48 by the ubiquitin-conjugating enzyme Cdc34

The attachment of ubiquitin (Ub) to lysines on substrates or itself by ubiquitin-conjugating (E2) and ubiquitin ligase (E3) enzymes results in protein ubiquitination. Lysine selection is important for generating diverse substrate-Ub structures and targeting proteins to different fates; however, the mechanisms of lysine selection are not clearly understood. The positioning of lysine(s) toward the E2/E3 active site and residues proximal to lysines are critical in their selection. We investigated determinants of lysine specificity of the ubiquitin-conjugating enzyme Cdc34, toward substrate and Ub lysines. Evaluation of the relative importance of different residues positioned −2, −1, +1 and +2 toward ubiquitination of its substrate, Sic1, on lysine 50 showed that charged residues in the −1 and −2 positions negatively impact on ubiquitination. Modeling suggests that charged residues at these positions alter the native salt-bridge interactions in Ub and Cdc34, resulting in misplacement of Sic1 lysine 50 in the Cdc34 catalytic cleft. During polyubiquitination, Cdc34 showed a strong preference for Ub lysine 48 (K48), with lower activity towards lysine 11 (K11) and lysine 63 (K63). Mutating the −2, −1, +1 and +2 sites surrounding K11 and K63 to mimic those surrounding K48 did not improve their ubiquitination, indicating that further determinants are important for Ub K48 specificity. Modeling the ternary structure of acceptor Ub with the Cdc34~Ub complex as well as in vitro ubiquitination assays unveiled the importance of K6 and Q62 of acceptor Ub for Ub K48 polyubiquitination. These findings provide molecular and structural insight into substrate lysine and Ub K48 specificity by Cdc34.     

Release of ubiquitin-charged Cdc34-S - Ub from the RING domain is essential for ubiquitination of the SCF(Cdc4)-bound substrate Sic1

The S. cerevisiae SCF(Cdc4) is a prototype of RING-type SCF E3s, which recruit substrates for polyubiquitination by the Cdc34 ubiquitin-conjugating enzyme. Current models propose that Cdc34 ubiquitinates the substrate while remaining bound to the RING domain. In contrast, we found that the formation of a ubiquitin thiol ester regulates the Cdc34/SCF(Cdc4) binding equilibrium by increasing the dissociation rate constant, with only a minor effect on the association rate. By using a F72VCdc34 mutant with increased affinity for the RING domain, we demonstrate that release of ubiquitin-charged Cdc34-S - Ub from the RING is essential for ubiquitination of the SCF(Cdc4)-bound substrate Sic1. Release of ubiquitin-charged E2 from E3 prior to ubiquitin transfer is a previously unrecognized step in ubiquitination, which can explain both the modification of multiple lysines on the recruited substrate and the extension of polyubiquitin chains. We discuss implications of this finding for function of other ubiquitin ligases.     

Mechanism of lysine 48-linked ubiquitin-chain synthesis by the cullin-RING ubiquitin-ligase complex SCF-Cdc34

Ubiquitin chains linked via lysine 48 (K48) of ubiquitin mediate recognition of ubiquitinated proteins by the proteasome. However, the mechanisms underlying polymerization of this targeting signal on a substrate are unknown. Here we dissect this process using the cyclin-dependent kinase inhibitor Sic1 and its ubiquitination by the cullin-RING ubiquitin ligase SCF(Cdc4) and the ubiquitin-conjugating enzyme Cdc34. We show that Sic1 ubiquitination can be separated into two steps: attachment of the first ubiquitin, which is rate limiting, followed by rapid elongation of a K48-linked ubiquitin chain. Mutation of an acidic loop conserved among Cdc34 orthologs has no effect on attachment of the first ubiquitin onto Sic1 but compromises the processivity and linkage specificity of ubiquitin-chain synthesis. We propose that the acidic loop favorably positions K48 of a substrate-linked ubiquitin to attack SCF bound Cdc34 approximately ubiquitin thioester and thereby enables processive synthesis of K48-linked ubiquitin chains by SCF-Cdc34.     

U box proteins as a new family of ubiquitin-protein ligases.

The U box is a domain of approximately 70 amino acids that is present in proteins from yeast to humans. The prototype U box protein, yeast Ufd2, was identified as a ubiquitin chain assembly factor that cooperates with a ubiquitin-activating enzyme (E1), a ubiquitin-conjugating enzyme (E2), and a ubiquitin-protein ligase (E3) to catalyze ubiquitin chain formation on artificial substrates. E3 enzymes are thought to determine the substrate specificity of ubiquitination and have been classified into two families, the HECT and RING finger families. Six mammalian U box proteins have now been shown to mediate polyubiquitination in the presence of E1 and E2 and in the absence of E3. These U box proteins exhibited different specificities for E2 enzymes in this reaction. Deletion of the U box or mutation of conserved amino acids within it abolished ubiquitination activity. Some U box proteins catalyzed polyubiquitination by targeting lysine residues of ubiquitin other than lysine 48, which is utilized by HECT and RING finger E3 enzymes for polyubiquitination that serves as a signal for proteolysis by the 26 S proteasome. These data suggest that U box proteins constitute a third family of E3 enzymes and that E4 activity may reflect a specialized type of E3 activity.     

Evaluation of a diffusion-driven mechanism for substrate ubiquitination by the SCF-Cdc34 ubiquitin ligase complex

Release of ubiquitin-charged Cdc34 from the SCF ubiquitin ligase followed by diffusion-driven collision with substrate has been proposed to underlie ubiquitination of the canonical SCF substrate Sic1. Cdc34 F72V, reported to be defective in dissociation from SCF, served as key validation. Here, we test predictions of this "hit-and-run" hypothesis. We find that Cdc34 F72V is generally defective in SCF-mediated activation but, contrary to expectation, does not compete with wild-type Cdc34 in vitro or in vivo and can fulfill the physiological role of Cdc34 with only moderate delay in Sic1 turnover. Whereas a hit-and-run mechanism might explain how Cdc34 can transfer ubiquitin to the ends of growing ubiquitin chains on SCF-bound substrates, molecular modeling suggests that an E2 docked to SCF can do so without dissociating. We propose that interactions between Cdc34 approximately Ub and SCF directly activate ubiquitin transfer within a substrate-SCF-Cdc34 approximately Ub ternary complex.     

Human Cdc34 employs distinct sites to coordinate attachment of ubiquitin to a substrate and assembly of polyubiquitin chains

The Cdc34 E2 ubiquitin (Ub) conjugating enzyme catalyzes polyubiquitination of a substrate recruited by the Skp1-Cullin 1-F-box protein-ROC1 E3 Ub ligase. Using mutagenesis studies, we now show that human Cdc34 employs distinct sites to coordinate the transfer of Ub to a substrate and the assembly of polyubiquitin chains. Mutational disruption of the conserved charged stretch (residues 143 to 153) or the acidic loop residues D102 and D103 led to accumulation of monoubiquitinated IkappaBalpha while failing to yield polyubiquitin chains, due to a catalytic defect in Ub-Ub ligation. These results suggest an ability of human Cdc34 to position the attacking Ub for assembly of polyubiquitin chains. Analysis of Cdc34N85Q and Cdc34S138A revealed severe defects of these mutants in both poly- and monoubiquitination of IkappaBalpha, supporting a role for N85 in stabilizing the oxyanion and in coordinating, along with S138, the attacking lysine for catalysis. Finally, Cdc34S95D and Cdc34(E108A/E112A) abolished both poly- and monoubiquitination of IkappaBalpha. Unexpectedly, the catalytic defects of these mutants in di-Ub synthesis can be rescued by fusion of a glutathione S-transferase moiety at E2's N terminus. These findings support the hypothesis that human Cdc34 S95 and E108/E112 are required to position the donor Ub optimally for catalysis, in a manner that might depend on E2 dimerization.     

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

Cdc34 is an E2 ubiquitin-conjugating enzyme that functions in conjunction with SCF (Skp1·Cullin 1·F-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 Lys⁴⁸ and C-terminal tail, both of which are key sites for conjugation. When bound to ubiquitin in one orientation, the Cdc34 UBS1 aromatic residues (Phe²⁰⁶, Tyr²⁰⁷, Tyr²¹⁰, and Tyr²¹¹) are probably positioned in the vicinity of ubiquitin C-terminal residue Val⁷⁰. Replacement of UBS1 aromatic residues by glycine or of ubiquitin Val⁷⁰ by alanine decreased UBS1-ubiquitin affinity interactions. UBS1 appeared to support the function of Cdc34 in vivo because human Cdc34(1–215) but not Cdc34(1–200) was able to complement the growth defect by yeast Cdc34 mutant strain. Finally, reconstituted IκBα ubiquitination analysis revealed a role for each adjacent pair of UBS1 aromatic residues (Phe²⁰⁶/Tyr²⁰⁷, Tyr²¹⁰/Tyr²¹¹) in conjugation, with Tyr²¹⁰ exhibiting the most pronounced catalytic function. Intriguingly, Cdc34 Tyr²¹⁰ was required for the transfer of the donor ubiquitin to a receptor lysine on either IκBα or a ubiquitin in a manner that depended on the neddylated RING sub-complex of the SCF. Taken together, our results identified a new ubiquitin binding activity within the human Cdc34 C terminus that contributes to SCF-dependent ubiquitination.     

The role of atypical ubiquitination in cell regulation

Ubiquitination is a type of intracellular proteins post-translational modification (PTM) characterized by covalent attachment of ubiquitin molecules to target proteins. This includes monoubiquitination (attachment of one ubiquitin molecule), multiple monoubiquitination also known as multiubiquitination (attachment of several monomeric ubiquitin molecules to a target protein), and polyubiquitination (attachment of ubiquitin chains consisting of several, most frequently four ubiquitin monomers to a target protein). In the case of polyubiquitination, linear or branched polyubiquitin chains are formed. Their formation involves various lysine residues of monomeric ubiquitin. The best studied is Lys48-linked polyubiquitination, which targets proteins for proteasomal degradation. In this review we have considered examples of so-called atypical polyubiquitination, which mainly involves other lysine residues (Lys6, Lys11, Lys27, Lys29, Lys33, Lys63) and also N-terminal methionine. The considered examples convincingly demonstrate that polyubiquitination of proteins (not necessarily) targets proteins for their proteolytic degradation in proteasomes. Atypically polyubiquitinated proteins are involved in regulation of various processes including immune response, genome stability, signal transduction, etc. Alterations of ubiquitination machinery is crucial for development of serious diseases.     

Protein monoubiquitination and polyubiquitination generate structural diversity to control distinct biological processes

Ubiquitination involves the attachment of ubiquitin (Ub) to lysine residues on substrate proteins or itself, which can result in protein monoubiquitination or polyubiquitination. Polyubiquitination through different lysines (seven) or the N-terminus of Ub can generate different protein-Ub structures. These include monoubiquitinated proteins, polyubiqutinated proteins with homotypic chains through a particular lysine on Ub or mixed polyubiquitin chains generated by polymerization through different Ub lysines. The ability of the ubiquitination pathway to generate different protein-Ub structures provides versatility of this pathway to target proteins to different fates. Protein ubiquitination is catalyzed by Ub-conjugating and Ub-ligase enzymes, with different combinations of these enzymes specifying the type of Ub modification on protein substrates. How Ub-conjugating and Ub-ligase enzymes generate this structural diversity is not clearly understood. In the current review, we discuss mechanisms utilized by the Ub-conjugating and Ub-ligase enzymes to generate structural diversity during protein ubiquitination, with a focus on recent mechanistic insights into protein monoubiquitination and polyubiquitination.     

Cdc34 C-terminal tail phosphorylation regulates Skp1/cullin/F-box (SCF)-mediated ubiquitination and cell cycle progression

The ubiquitin-conjugating enzyme Cdc34 (cell division cycle 34) plays an essential role in promoting the G1-S-phase transition of the eukaryotic cell cycle and is phosphorylated in vivo. In the present study, we investigated if phosphorylation regulates Cdc34 function. We mapped the in vivo phosphorylation sites on budding yeast Cdc34 (yCdc34; Ser207 and Ser216) and human Cdc34 (hCdc34 Ser203, Ser222 and Ser231) to serine residues in the acidic tail domain, a region that is critical for Cdc34's cell cycle function. CK2 (protein kinase CK2) phosphorylates both yCdc34 and hCdc34 on these sites in vitro. CK2-mediated phosphorylation increased yCdc34 ubiquitination activity towards the yeast Saccharomyces cerevisiae Sic1 in vitro, when assayed in the presence of its cognate SCFCdc4 E3 ligase [where SCF is Skp1 (S-phase kinase-associated protein 1)/cullin/F-box]. Similarly, mutation of the yCdc34 phosphorylation sites to alanine, aspartate or glutamate residues altered Cdc34-SCFCdc4-mediated Sic1 ubiquitination activity. Similar results were obtained when yCdc34's ubiquitination activity was assayed in the absence of SCFCdc4, indicating that phosphorylation regulates the intrinsic catalytic activity of Cdc34. To evaluate the in vivo consequences of altered Cdc34 activity, wild-type yCdc34 and the phosphosite mutants were introduced into an S. cerevisiae cdc34 deletion strain and, following synchronization in G1-phase, progression through the cell cycle was monitored. Consistent with the increased ubiquitination activity in vitro, cells expressing the phosphosite mutants with higher catalytic activity exhibited accelerated cell cycle progression and Sic1 degradation. These studies demonstrate that CK2-mediated phosphorylation of Cdc34 on the acidic tail domain stimulates Cdc34-SCFCdc4 ubiquitination activity and cell cycle progression.     

Structural insights into NEDD8 activation of cullin-RING ligases: conformational control of conjugation

Cullin-RING ligases (CRLs) comprise the largest ubiquitin E3 subclass, in which a central cullin subunit links a substrate-binding adaptor with an E2-binding RING. Covalent attachment of the ubiquitin-like protein NEDD8 to a conserved C-terminal domain (ctd) lysine stimulates CRL ubiquitination activity and prevents binding of the inhibitor CAND1. Here we report striking conformational rearrangements in the crystal structure of NEDD8~Cul5(ctd)-Rbx1 and SAXS analysis of NEDD8~Cul1(ctd)-Rbx1 relative to their unmodified counterparts. In NEDD8ylated CRL structures, the cullin WHB and Rbx1 RING subdomains are dramatically reoriented, eliminating a CAND1-binding site and imparting multiple potential catalytic geometries to an associated E2. Biochemical analyses indicate that the structural malleability is important for both CRL NEDD8ylation and subsequent ubiquitination activities. Thus, our results point to a conformational control of CRL activity, with ligation of NEDD8 shifting equilibria to disfavor inactive CAND1-bound closed architectures, and favor dynamic, open forms that promote polyubiquitination.     

Oxidative Stress Responses Involve Oxidation of a Conserved Ubiquitin Pathway Enzyme

Although it is vital that cells detect and respond to oxidative stress to allow adaptation and repair damage, the underlying sensing and signaling mechanisms that control these responses are unclear. Protein ubiquitinylation plays an important role in controlling many biological processes, including cell division. In Saccharomyces cerevisiae, ubiquitinylation involves a single E1 enzyme, Uba1, with multiple E2s and E3s providing substrate specificity. For instance, the conserved E2 Cdc34 ubiquitinylates many substrates, including the cyclin-dependent kinase inhibitor Sic1, targeting it for degradation to allow cell cycle progression. Here we reveal that, in contrast to other ubiquitin pathway E2 enzymes, Cdc34 is particularly sensitive to oxidative inactivation, through sequestration of the catalytic cysteine in a disulfide complex with Uba1, by levels of oxidant that do not reduce global ubiquitinylation of proteins. This Cdc34 oxidation is associated with (i) reduced levels of Cdc34-ubiquitin thioester forms, (ii) increased stability of at least one Cdc34 substrate, Sic1, and (iii) Sic1-dependent delay in cell cycle progression. Together, these data reveal that the differential sensitivity of a ubiquitin pathway E2 enzyme to oxidation is utilized as a stress-sensing mechanism to respond to oxidative stress.     

Biochemical and Proteomic Analysis of Ubiquitination of Hsc70 and Hsp70 by the E3 Ligase CHIP

The E3 ubiquitin ligase CHIP is involved in protein triage, serving as a co-chaperone for refolding as well as catalyzing ubiquitination of substrates. CHIP functions with both the stress induced Hsp70 and constitutive Hsc70 chaperones, and also plays a role in maintaining their balance in the cell. When the chaperones carry no client proteins, CHIP catalyzes their polyubiquitination and subsequent proteasomal degradation. Although Hsp70 and Hsc70 are highly homologous in sequence and similar in structure, CHIP mediated ubiquitination promotes degradation of Hsp70 with a higher efficiency than for Hsc70. Here we report a detailed and systematic investigation to characterize if there are significant differences in the CHIP in vitro ubiquitination of human Hsp70 and Hsc70. Proteomic analysis by mass spectrometry revealed that only 12 of 39 detectable lysine residues were ubiquitinated by UbcH5a in Hsp70 and only 16 of 45 in Hsc70. The only conserved lysine identified as ubiquitinated in one but not the other heat shock protein was K159 in Hsc70. Ubiquitination assays with K-R ubiquitin mutants showed that multiple Ub chain types are formed and that the distribution is different for Hsp70 versus Hsc70. CHIP ubiquitination with the E2 enzyme Ube2W is predominantly directed to the N-terminal amine of the substrate; however, some internal lysine modifications were also detected. Together, our results provide a detailed view of the differences in CHIP ubiquitination of these two very similar proteins, and show a clear example where substantial differences in ubiquitination can be generated by a single E3 ligase in response to not only different E2 enzymes but subtle differences in the substrate.     

Structure of the HHARI Catalytic Domain Shows Glimpses of a HECT E3 Ligase

The ubiquitin-signaling pathway utilizes E1 activating, E2 conjugating, and E3 ligase enzymes to sequentially transfer the small modifier protein ubiquitin to a substrate protein. During the last step of this cascade different types of E3 ligases either act as scaffolds to recruit an E2 enzyme and substrate (RING), or form an ubiquitin-thioester intermediate prior to transferring ubiquitin to a substrate (HECT). The RING-inBetweenRING-RING (RBR) proteins constitute a unique group of E3 ubiquitin ligases that includes the Human Homologue of Drosophila Ariadne (HHARI). These E3 ligases are proposed to use a hybrid RING/HECT mechanism whereby the enzyme uses facets of both the RING and HECT enzymes to transfer ubiquitin to a substrate. We now present the solution structure of the HHARI RING2 domain, the key portion of this E3 ligase required for the RING/HECT hybrid mechanism. The structure shows the domain possesses two Zn²⁺-binding sites and a single exposed cysteine used for ubiquitin catalysis. A structural comparison of the RING2 domain with the HECT E3 ligase NEDD4 reveals a near mirror image of the cysteine and histidine residues in the catalytic site. Further, a tandem pair of aromatic residues exists near the C-terminus of the HHARI RING2 domain that is conserved in other RBR E3 ligases. One of these aromatic residues is remotely located from the catalytic site that is reminiscent of the location found in HECT E3 enzymes where it is used for ubiquitin catalysis. These observations provide an initial structural rationale for the RING/HECT hybrid mechanism for ubiquitination used by the RBR E3 ligases.     

E3 ligases determine ubiquitination site and conjugate type by enforcing specificity on E2 enzymes

Ubiquitin-conjugating enzymes (E2s) have a dominant role in determining which of the seven lysine residues of ubiquitin is used for polyubiquitination. Here we show that tethering of a substrate to an E2 enzyme in the absence of an E3 ubiquitin ligase is sufficient to promote its ubiquitination, whereas the type of the ubiquitin conjugates and the identity of the target lysine on the substrate are promiscuous. In contrast, when an E3 enzyme is introduced, a clear decision between mono- and polyubiquitination is made, and the conjugation type as well as the identity of the target lysine residue on the substrate becomes highly specific. These features of the E3 can be further regulated by auxiliary factors as exemplified by MDMX (Murine Double Minute X). In fact, we show that this interactor reconfigures MDM2-dependent ubiquitination of p53. Based on several model systems, we propose that although interaction with an E2 is sufficient to promote substrate ubiquitination the E3 molds the reaction into a specific, physiologically relevant protein modification.     

The loop-less tmCdc34 E2 mutant defective in polyubiquitination in vitro and in vivo supports yeast growth in a manner dependent on Ubp14 and Cka2

Background

The S73/S97/loop motif is a hallmark of the Cdc34 family of E2 ubiquitin-conjugating enzymes that together with the SCF E3 ubiquitin ligases promote degradation of proteins involved in cell cycle and growth regulation. The inability of the loop-less ^Δ12Cdc34 mutant to support growth was linked to its inability to catalyze polyubiquitination. However, the loop-less triple mutant (tm) Cdc34, which not only lacks the loop but also contains the S73K and S97D substitutions typical of the K73/D97/no loop motif present in other E2s, supports growth. Whether ^tmCdc34 supports growth despite defective polyubiquitination, or the S73K and S97D substitutions, directly or indirectly, correct the defect caused by the loop absence, are unknown.

Results

^tmCdc34 supports yeast viability with normal cell size and cell cycle profile despite producing fewer polyubiquitin conjugates in vivo and in vitro. The in vitro defect in Sic1 substrate polyubiquitination is similar to the defect observed in reactions with ^Δ12Cdc34 that cannot support growth. The synthesis of free polyubiquitin by ^tmCdc34 is activated only modestly and in a manner dependent on substrate recruitment to SCF^Cdc4. Phosphorylation of C-terminal serines in ^tmCdc34 by Cka2 kinase prevents the synthesis of free polyubiquitin chains, likely by promoting their attachment to substrate. Nevertheless, ^tm CDC34 yeast are sensitive to loss of the Ubp14 C-terminal ubiquitin hydrolase and DUBs other than Ubp14 inefficiently disassemble polyubiquitin chains produced in ^tm CDC34 yeast extracts, suggesting that the free chains, either synthesized de novo or recycled from substrates, have an altered structure.

Conclusions

The catalytic motif replacement compromises polyubiquitination activity of Cdc34 and alters its regulation in vitro and in vivo, but either motif can support Cdc34 function in yeast viability. Robust polyubiquitination mediated by the S73/S97/loop motif is thus not necessary for Cdc34 role in yeast viability, at least under typical laboratory conditions.     

The Acidic Tail domain of Human Cdc34 is Required for p27Kip1 Ubiquitination and Complementation of a cdc34 Temperature Sensitive Yeast Strain

Human Cdc34 is an ubiquitin conjugating enzyme or E2 that ubiquitinates substrates including p27Kip1, I?B?, Wee1, and MyoD. Cdc34 possesses a core catalytic domain encoding the active site cysteine and an acidic tail domain within the carboxyl terminal 36 amino acids. Studies suggest that Cdc34 is phosphorylated in mammalian cells at 5 potential residues within the tail domain. In order to study the biological significance of the Cdc34 acidic tail domain and the possible significance of phosphorylation within this region, we tested the ability of human Cdc34 mutants to complement the cdc34-2 temperature sensitive (ts) strain of Saccharomyces cerevisiae. Our studies indicated that complementation of the cdc34-2 ts strain was critically dependent upon the carboxyl-terminal 36 amino acids of human Cdc34, but did not require phosphorylation of human Cdc34 residues S203, S222, S231, T233, and S236. Further studies demonstrated that although a Cdc34 mutant bearing a deletion of the C-terminal 36 amino acids (Cdc34 1-200) was efficiently charged with ubiquitin by E1, it was severely reduced for the ability to ubiquitinate p27Kip1 in vitro compared to wildtype Cdc34. Both in vivo and in vitro binding studies indicated that Cdc34 1-200 bound to the E3-SCF components, Cul1 and Roc1, at levels comparable to the wildtype Cdc34. These studies suggest that the 36 amino acid acidic tail domain of human Cdc34 is critical for its ability to transfer ubiquitin to a substrate and is dispensable for the association of Cdc34 with Cul1 and Roc1. We postulate that the tail domain of Cdc34 may be important for its efficient dissociation from Cul1 and Roc1, an essential requirement for ubiquitination by the budding yeast Cdc34p, or it may be required more directly for ubiquitin transfer to the substrate.     

E2-BRCA1 RING interactions dictate synthesis of mono- or specific polyubiquitin chain linkages

An E3 ubiquitin ligase mediates the transfer of activated ubiquitin from an E2 ubiquitin-conjugating enzyme to its substrate lysine residues. Using a structure-based, yeast two-hybrid strategy, we discovered six previously unidentified interactions between the human heterodimeric RING E3 BRCA1-BARD1 and the human E2s UbcH6, Ube2e2, UbcM2, Ubc13, Ube2k and Ube2w. All six E2s bind directly to the BRCA1 RING motif and are active with BRCA1-BARD1 for autoubiquitination in vitro. Four of the E2s direct monoubiquitination of BRCA1. Ubc13-Mms2 and Ube2k direct the synthesis of Lys63- or Lys48-linked ubiquitin chains on BRCA1 and require an acceptor ubiquitin attached to BRCA1. Differences between the mono- and polyubiquitination activities of the BRCA1-interacting E2s correlate with their ability to bind ubiquitin noncovalently at a site distal to the active site. Thus, BRCA1 has the ability to direct the synthesis of specific polyubiquitin chain linkages, depending on the E2 bound to its RING.