Despite the importance of ubiquitination, the mechanisms that control the selection of lysines on substrates and Ub are poorly understood. Our studies demonstrate that compatibility between amino acids proximal to acceptor lysines and residues in the catalytic core of Cdc34 ultimately determines lysine ubiquitination in substrates and Ub. This new concept expands upon the current view that ubiquitination is dependent primarily on the proximity of the lysine to the E2-thioester mediated through positioning by E2/E3 (21
) and has important implications for our understanding of the mechanisms of substrate monoubiquitination and polyubiquitination.
Substrates such as Sic1, which are ubiquitinated on numerous lysines, indicate that mechanisms for selecting spatially separated lysines must exist. As described in the introduction, several higher-order structural mechanisms contribute to the flexibility in the positioning of different lysines for substrate ubiquitination, such as the E3-mediated positioning of lysines toward the E2~Ub thioester. Lysine selection flexibility may also be achieved by the release of the ubiquitin-charged Cdc34~Ub from SCF to transfer Ub to different substrate lysines, as proposed by the “hit-and-run” hypothesis (5
). Our studies now demonstrate that in addition to these higher-order effects, amino acids immediately proximal to lysines also play an important role in catalysis, suggesting that the interplay of both mechanisms ultimately defines the efficiency of the ubiquitination of a lysine. SCFCdc4
/Cdc34 displayed a clear preference toward Sic1 K53. The importance of the sequence environment was first exemplified by our studies mutating residues around Sic1 lysines, where changes to proximal amino acids consistently reduced or increased ubiquitination. Importantly, the growth rate of S. cerevisiae
expressing the different single-lysine Sic1 mutants or the K53 sequence mutants correlated with their level of ubiquitination in vitro
, indicating that sequence-dependent ubiquitination is physiologically important (Fig. and ). This was due to the altered stability of Sic1 in S. cerevisiae
, as exemplified by Sic1 K53 and Sic1 K32 and their derivatives. The importance of Sic1 lysine selectivity during ubiquitination is exemplified by studies demonstrating that the half-life of Sic1 ubiquitinated on K36 or K84 is 1 min or 5 min, respectively, indicating that the context of Sic1 lysine ubiquitination is important for proteolysis (20
In addition to Sic1 lysines, we observed that alterations around Ub K48 can affect polyubiquitination by Cdc34. Hence, catalysis was severely impaired with Ub(G47Q), while Ub(Q49P) and Ub(L50S) did not affect the utilization of K48 for polyubiquitination. Previous reports have shown that G47 of Ub is important for yeast cell growth and Ub conjugation via K48 (21
). Similar to the E3-mediated positioning of substrate lysines, structural aspects of some E2s, such as Ubc13 and Cdc34, position Ub lysines to generate Ub chains of a specific topology (11
). However, E2s such as human UbcH5 can utilize all Ub lysines but display a preference for K11, K48, and K63, indicating less structural constraint and that other mechanisms contribute to Ub lysine specificity (12
). Our work suggests that amino acids proximal to lysines in Ub may provide a further level of specificity control with certain E2s.
Importantly, our studies show that ubiquitination is linked to key residues in the catalytic core of Cdc34, which can differentially regulate initial substrate ubiquitination and Ub chain extension via K48. It is unclear if the divergence of residues surrounding the catalytic cysteine in different E2s is functionally important. Our results argue that this divergence may have evolved to contribute to lysine specificity in substrates and Ub. Previous studies underscored the importance of the analogous sites in other E2s. Studies of Ubc9 in complex with the substrate RanGAP1 demonstrated that Ubc9 N85, Y87, and D127 facilitate catalysis through the optimal alignment and nucleophilic activation of the attacking lysine within the active site (38
). The human Cdc34(Y87A) and Cdc34(S138A) mutants are impaired in IκBα ubiquitination (7
). The yeast Ubc13(D81K) mutant is completely inactive (35
). Our studies delineate their specific importance, showing that these residues are important for lysine selectivity and can specify if Sic1 is monoubiquitinated or polyubiquitinated. This was strikingly exemplified by the YD
A and N
SA mutants, which displayed polar-opposite activities toward lysines of Sic1 and K48 of Ub. Hence, the YD
A mutant was as active as Cdc34 in the ubiquitination of Sic1 but essentially inactive toward K48 of Ub. Conversely, the N
SA mutant was significantly impaired in Sic1 ubiquitination but active toward K48 of Ub (Fig. and and Table ). Therefore, these mutants display dichotomy and are not inactive per se
but rather in their specificity toward particular acceptor lysines. These studies strongly suggest that different combinations of residues in the catalytic regions of E2s are not functionally redundant. E2s may have evolved different active-site structures for the optimal ubiquitination of specific lysines in a particular range of substrates and possibly in dictating whether a substrate is mono- or polyubiquitinated. The regulatory nature of the site analogous to Cdc34 S139 is further illustrated in E2s such as hHR6A, which is phosphorylated by CDKs on serine at this position (S120) to regulate activity (27
), although not all E2s with serine in this position are phosphorylated at this site, e.g., Cdc34 (3
). By recruiting a particular E2 or E2s, E3 may specify the mode of substrate ubiquitination. In support of this notion, although E3s such as SCF and human APC/C use single E2s for attaching Ub to both substrate and Ub lysines, other E3s utilize different E2s for specific roles during polyubiquitination. Hence, yeast APC/C utilizes Ubc4 and Ubc1 for ubiquitinating cyclin B (23
). Ubc4 catalyzes cyclin B monoubiquitination, while Ubc1 catalyzes further K48-mediated polyubiquitination.
Our studies with the YD
A mutant highlighted the importance of compatibility between the lysine environment and E2 core residues. While this mutant was impaired in its ability to utilize Ub K48, changes to the proximal residues (Q49P and L50S) restored the activity of this mutant in Ub chain assembly (Fig. and ). Although the structure of yeast Cdc34 is not known, the catalytic domain of E2 enzymes is highly conserved (22
). Structure-function studies of the E2-substrate complex of human Ubc9 and RanGAP1 showed that the sites analogous to Cdc34 Y89, S139, and A141 (Ubc9 Y87, D127, and A129) surround the catalytic cysteine. Ubc9 Y87 and A129 make van der Waals contacts with L525, S527, and E528 of RanGAP1, which are proximal to the sumoylated K526, while D127 is within hydrogen-bonding distance of sumoylated K526 (2
). These interactions are important for sumoylation through optimal alignment and pK suppression for the nucleophilic activation of the attacking lysine (2
). Our studies suggest that similar contacts between the Cdc34 core sites and the ubiquitination site sequence are important for catalysis. Our kinetic studies suggest that compatibility is important for lysine activation rather than substrate binding. Alternatively, it is possible that some of the Cdc34 residues examined in this study may be important for facilitating Cdc34 activity via self-association, which is important for its catalytic activity (36
). However, these studies demonstrated that the active-site cysteine and serines 73 and 97, which are distinct from the sites evaluated in this study, are important for oligomerization. Future structural studies of the Cdc34-substrate complex will provide further insights into these issues.
In agreement with our findings, recent studies with UbcH10 and its cognate E3, APC/C, demonstrated that a sequence motif, termed the TEK box, surrounding lysines in the substrate and Ub K11 is important for ubiquitination (11
). In addition, proteins with Ub binding domains (UBDs) can be ubiquitinated directly by E2s without E3s (10
). It is postulated that the substrate UBD binds to the Ub conjugated to the E2 and that Ub is then transferred to the substrate lysine. Seven different E2s displayed differential specificity toward different UBD proteins, raising the issue of how a particular UBD substrate is selected for ubiquitination by a particular E2, since there is no E3 to direct specificity. Our studies suggest that the compatibility of the E2 catalytic core with residues surrounding potential target lysines is important for controlling specificity between particular E2s and UBD proteins.
Altogether, these findings demonstrate that in addition to the importance of higher-order structural features of SCFCdc4 and Cdc34 or models such as the “hit-and-run” hypothesis, which position different lysines for the attack of the Cdc34~Ub thioester bond, compatibility between key residues within the Cdc34 catalytic region and those proximal to acceptor lysines provides a further level of specificity control. E2s may have evolved divergent structures in their catalytic regions to modulate lysine specificity. It will be interesting to comprehensively define all the important determinants in the catalytic region of E2s and residues proximal to acceptor lysines to define if this is a general feature of E2-mediated ubiquitination.