Polyubiquitin chain assembly occurs in two steps. First, one of many lysines on a substrate attacks an E2-ubiquitin thioester, resulting in transfer of ubiquitin to the lysine (monoubiquitination). In the case of the yeast APC, this first reaction is catalyzed most efficiently by Ubc4, suggesting that the Ubc4 active site is specialized for recognition of lysines that are typically found in a disordered peptide context (Rodrigo-Brenni and Morgan, 2007
). Second, a specific lysine in the attached ubiquitin attacks another E2-ubiquitin thioester to initiate assembly of a polyubiquitin chain. In yeast APC reactions, this second step is catalyzed by Ubc1, the active site of which is specialized for recognition of K48 in the globular ubiquitin protein.
Our results demonstrate that the K48 specificity of Ubc1 depends on a group of residues on two loops lying next to each other near the active-site cysteine (, S2
). Single mutations of key residues in this group (T84, Q122, and A124) result in major defects in K48-specific ubiquitin attack. We propose that the side chains of these residues form a landing pad that globally orients the incoming ubiquitin for efficient K48-specific attack of the Ubc1-ubiquitin thioester.
The mutations we analyzed in detail were found to reduce K48-specific catalytic rate but not ubiquitin binding affinity, as judged by the similar Kd values obtained in diubiquitin synthesis assays. Thus, we did not identify a mutation that significantly disrupts ubiquitin binding, perhaps indicating that the interacting surface depends on multiple low affinity interactions involving several residues.
The high Kd
values we observed in our diubiquitin synthesis reactions are similar to those observed with other E2s (Petroski and Deshaies, 2005
), suggesting that the affinity of the attacking ubiquitin for an E2-ubiquitin conjugate is very low. This interaction, in the 350 μM range, would not be expected to result in significant reaction rates at the low concentrations of ubiquitin (~10 μM) inside the cell. Productive attack of Ubc1 occurs only at significant rates in the context of the E3, where binding of the substrate near the E2 would greatly increase its effective concentration and thereby drive the reaction. In addition, the binding of the E3 is thought to enhance the catalytic activity of the E2 (Ozkan et al., 2005
; Petroski and Deshaies, 2005
), which would increase kcat
even at low substrate occupancy.
Our results with the diubiquitin synthesis assay also indicate that the binding of ubiquitin to the Ubc1 active site, as reflected in apparent Kd
, does not depend on the C-terminal UBA domain. This domain has ubiquitin-binding activity, but its function remains unclear. As deletion of the UBA domain reduces the processivity of chain assembly by the APC (Rodrigo-Brenni and Morgan, 2007
), one possibility is that the UBA domain increases the affinity of Ubc1 for APC-bound substrates carrying multiple ubiquitins.
Much of our work focused on T84, which we found is required specifically for K48-dependent catalysis. Mutation of this residue abolished K48-linked chain formation without affecting the slow rate of ubiquitination of non-K48 lysines. The T84G mutant appears to display normal affinity for ubiquitin but has a major catalytic defect. Based on the normal pH dependency of the reaction with T84G, we conclude that T84 does not help suppress the pKa
of the attacking lysine. In the tertiary structure of Ubc1, the T84 side chain is exposed on a surface loop and appears disordered in the NMR ensemble (Merkley and Shaw, 2004
) (). Mutation of T84 is not likely to change the conformation of this loop or nearby side chains in Ubc1. In addition, the T84 side chain is about 12 Å from the active-site cysteine of Ubc1, which is too distant for it to be directly involved in positioning K48 during the reaction. Interestingly, T84 is located in a position similar to that of S89 in Ubc9, which helps orient the substrate by interacting with a glutamate near the attacking lysine (, bottom). We therefore suspect that T84 influences catalytic rate through indirect or global effects on K48 positioning, perhaps through interactions between the T84 hydroxyl and residues in ubiquitin other than K48.
We found that mutation of A124 to a proline (as in Ubc4) also resulted in a significant defect in K48-specific ubiquitination. The placement of a proline at this position, at the junction of a loop and an alpha helix (), might be expected to generate local conformational changes that influence the positions of nearby residues, such as T84 or Q122.
Q122 is another important residue in K48-specific ubiquitination. Mutation of this residue to leucine, as in Ubc4, resulted in a 4-fold drop in the rate of K48-specific diubiquitin synthesis, and mutation to alanine or other amino acids greatly reduced K48-specific chain formation. As in the case of T84G, the Q122L and Q122A mutations affected catalytic rate and not ubiquitin binding affinity. Like the side chain of T84, that of Q122 is exposed and flexible on an ordered surface loop (), and mutations at this site are not expected to cause local conformational changes in the protein backbone. A comparison of Ubc1 structure with that of Ubc9 () suggests that Q122 is well positioned to influence the orientation of the attacking lysine, either directly or indirectly through interactions with nearby residues on ubiquitin.
Interestingly, the decreased K48-specific activity of the Q122L mutant was accompanied by an increase in its activity toward non-K48 lysines on the cyclin substrate. This change in behavior was particularly dramatic in studies of the attack of Ubc1 by cyclin substrate in the absence of the APC. Wild-type Ubc1 ubiquitinated lysines on the cyclin substrate very poorly, whereas Ubc1-Q122L displayed 60-fold higher activity toward cyclin. Thus, a leucine at this position greatly enhances the ubiquitination of lysines in the cyclin substrate. The glutamine that is normally at this position in Ubc1 limits its activity toward these lysines.
Our results with the Q122L mutant are reminiscent of previous studies of the SUMO E2, Ubc9 (Yunus and Lima, 2006
). Ubc9 contains a tyrosine, Y87, that contributes to a hydrophobic microenvironment that helps position the attacking lysine and suppress its pKa
(, bottom). A tyrosine is found at this position in several E2s (Yunus and Lima, 2006
). Many other E2s, however, do not have this tyrosine but instead contain a leucine, at a different position, that seems to fulfil the tyrosine's role. Ubc4 is a member of this latter group: indeed, it is this leucine of Ubc4 (L120) that is changed to a glutamine (Q122) in Ubc1 (Figure S2
). Thus, it seems likely that this leucine in Ubc4 helps catalyze non-K48 lysine ubiquitination. Ubc1, however, is unique among yeast E2s in not containing either the tyrosine (which is S81 in Ubc1) or the leucine (Q122). Furthermore, our pH dependency studies suggest that the lysine pKa
is not suppressed by Ubc1 as much as it is in Ubc9 reactions. We therefore speculate that deprotonation and positioning of the attacking K48 side chain involves mechanisms that are at least partly distinct from those employed by Ubc9.
We also explored the residues in ubiquitin itself that are important for K48-linked chain formation. Mutation of numerous residues adjacent to K48 of ubiquitin had little effect, but K48-specific activity was severely inhibited by mutation of Y59 to alanine or small hydrophobic residues. Activity was not greatly affected by mutation to phenylalanine and only partly inhibited by mutation to histidine, arguing that the ring structure of Y59, and not simply its hydrophobicity, is critical for activity. Interestingly, iodination of Y59 reduces ubiquitin chain assembly by E2-25K, the human ortholog of Ubc1 (Pickart et al., 1992
), indicating that the function of the Y59 ring is disrupted by iodination.
Detailed analysis of the Y59L ubiquitin mutant revealed a severe reduction of catalytic rate, with little effect on affinity for Ubc1. Y59 is therefore required for the catalysis of K48-specific ubiquitination, providing a potential example of substrate-assisted catalysis. In the tertiary structure of ubiquitin, Y59 does not contact the K48 side chain but could partly limit its movement. The Y59 side chain is partially buried and interacts with the hydrophobic protein core (notably L50), and the reduced heat stability we observed with some Y59 mutants might have resulted from local packing defects. The Y59L mutant, on the other hand, is nearly as heat-stable as wild-type ubiquitin but still displays a major catalytic defect. We suspect that the ring of Y59 in ubiquitin, like the hydroxyl of T84 in Ubc1, helps orient ubiquitin for productive attack of the E2. Indeed, Y59 and T84 might depend on each other for a common orientation function, which would explain our observation that mutation of either residue (or the two mutations in combination) abolishes almost all K48-specific activity. Another intriguing possibility arises from the fact, mentioned above, that Ubc1 is unusual among E2s because it does not contain the tyrosine or leucine that helps form a hydrophobic microenvironment around the attacking lysine. Perhaps Y59 on the ubiquitin substrate helps fulfil this role.
Numerous lines of evidence suggest that the K48 specificity of Ubc1 depends on mechanisms that are distinct from those used by another K48-specific E2, Cdc34. First, our work shows that Ubc1 activity toward K48 depends on a polar cluster of residues that are found near the cysteine of the Ubc1 active site but are not found in Cdc34; on the other hand, the K48 specificity of Cdc34 depends on an acidic loop, not found in Ubc1, that is inserted in the active site (Figure S2
) (Petroski and Deshaies, 2005
). Second, we found that Ubc1 activity was unaffected by mutation of I44 in the ubiquitin hydrophobic patch, whereas this mutation greatly reduces Cdc34 activity. Finally, iodination of Y59 was shown previously to inhibit activity with E2-25K, as mentioned above, but did not affect activity with the human Cdc34 ortholog (Pickart et al., 1992
). We are left with the remarkable conclusion that two enzymes have evolved distinct mechanisms to catalyze K48-specific polyubiquitin assembly.
It is becoming clear that the E2 active site contains residues that use varied and poorly understood mechanisms to catalyze lysine attack. Moreover, the active sites of different E2 proteins contain unique combinations of residues that confer specificity for lysines in distinct contexts. General E2s, such as Ubc4, carry residues that promote effective attack by a lysine in a polypeptide context that appears nonspecific and disordered. Other E2s (e.g. Ubc1, Ubc13, and Cdc34) carry active-site residues that direct enzyme activity toward specific lysines, such as K48 or K63, within the globular context of ubiquitin. In addition, the ubiquitin substrate itself contributes residues that help determine lysine specificity.