From Loops to Chains: Unraveling the Mysteries of Polyubiquitin Chain Specificity and Processivity Mathew E. Sowa and J. Wade Harper* Department of Pathology, Harvard Medical School, 77 Avenue Louis Pasteur, Boston, Massachusetts 02115
A B S T R A C T Regulated protein degradation via polyubiquitination controls almost every aspect of eukaryotic cellular biology; however, the precise mechanism by which specifically linked polyubiquitin chains are formed on target proteins as well as how the processivity of chain elongation is achieved remains a mystery. Recent work using the yeast ubiquitin ligase SCFCdc4 and the ubiquitin conjugating enzyme, Cdc34, has helped to answer these questions by identifying the determinants of lysine-48 specific ubiquitin chain polymerization.
*To whom correspondence should be addressed. E-mail: wade_harper@hms. harvard.edu.
Published online February 17, 2006 10.1021/cb0600020 CCC: $33.50 © 2006 by American Chemical Society
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rotein turnover through the ubiquitin–proteasome pathway controls a vast assortment of signaling pathways, and genetic inactivation of individual components in the system is responsible for numerous human diseases, ranging from neurodegenerative diseases to cancer (1, 2 ). In response to particular signals, specific regulatory proteins are tagged with a chain of ubiquitin molecules through the action of an enzymatic cascade composed of an E1 ubiquitin activating enzyme, an E2 ubiquitin conjugating enzyme (Ubc), and an E3 ubiquitin ligase. E3s are responsible for binding substrates and for bringing substrates into the proximity of a ubiquitin-charged E2, which then transfers ubiquitin to the substrate and to the ensuing ubiquitin chain in a processive reaction. Formation of lysine-48 linked polyubiquitin chains facilitates recognition and degradation by the 26S proteasome (1 ). During the last decade, our understanding of the genes that contribute to the ubiquitin pathway and many of the rules that control substrate selection by E3s have been uncovered. In addition, structural information is available for each of the two main classes of E3s: HECT and RING domain containing proteins (3, 4 ). These structures reveal the major role of proximity in facilitating ubiquitin transfer to substrates but tell us very little about the dynamics and chemical mechanisms involved in ubiquitin transfer and chain elongation. Indeed, a central unanswered question concerns how particular lysine residues in either the substrate or the growing ubiquitin chain are selected for chain elongation. Two
major classes of ubiquitin–ubiquitin linkages, lysine-48 (K48) and lysine-63 (K63), are found in vivo, but precisely how E2s select one of the seven lysines in ubiquitin (Figure 1, panel a) over another is poorly defined. Moreover, ubiquitination reactions can be highly processive, yet it is unclear how processivity is achieved given the structural constraints of a growing ubiquitin chain. Recent work published by Petroski and Deshaies (5) has provided novel insight into how the yeast E2, Cdc34, functions together with the RING E3, SCFCdc4, to promote processive polyubiquitination of its substrate, Sic1, and how selective chain elongation at K48 in ubiquitin is achieved. Unexpectedly, acceleration of the ubiquitin–ubiquitin conjugation step appears to reflect a specialized motif in Cdc34 that serves to orient K48 in the acceptor ubiquitin molecule for attack on the donor ubiquitin thiol-ester bond. Conjugating Ubiquitin. Ubiquitin is a 76 amino acid protein (Figure 1, panel a) that becomes activated for conjugation through the action of E1 ubiquitin activating enzyme. E1 uses ATP to form a thiol-ester between its active site cysteine residue and the C-terminal carboxylate of ubiquitin. The E1-S~Ub intermediate is then recognized by one of more than 30 E2 ubiquitin conjugating enzymes encoded by the human genome, which use an active site cysteine to perform a trans thiolesterification reaction giving E2-S~Ub. At steady state in vivo, individual E2s exist as a mixture of ubiquitin-charged and -uncharged forms. E2 enzymes possess a highly conserved core domain, containw w w. a c s c h e m i ca l biology.org
Figure 1. Structure of ubiquitin and the E2. a) Ubiquitin is small protein comprised of only 76 amino acids and forms a compact structure with the C-terminal tail exposed. A glycine residue is found at the C-terminus, which is used to form the thiol-ester bond between ubiquitin and the active site cysteine in both E1 and E2 proteins and which ultimately makes an isopeptide linkage with either the substrate or with a second ubiquitin molecule. Ubiquitin has seven surface-exposed lysine residues, the most well studied of which are K48 and K63. b) Two different E2 proteins, S. cerevisiae Ubc7 (2UCV) and H. sapiens UbcH5 (1ESK), are shown aligned by the Cα backbones using Pymol. The acidic loop insertion in Ubc7 is clearly visible and is located near the E3 binding site. I88 from UbcH5 is found on the 310 helix common to all E2s.
ing the active site cysteine, as well as sequences that are used to recognize RING or HECT motifs in E3s (Figure 1, panel b). Many E2s have yet to be studied in detail, but it is clear that there is some specificity among E2s for particular subclasses of E3s. However, the rules dictating the functional complementarity of E2s and E3s are largely unknown. Once a particular ubiquitin-charged E2 associates with a RING or HECT domain present in an E3, one of two processes can occur. If the E3 lacks a bound substrate, the E2 can discharge its ubiquitin to form an isopeptide bond with the ε-amino group from one of seven lysine residues in a second ubiquitin molecule through a poorly understood process. Indeed, for some isolated RING domain E3s in the absence of substrate, binding to the E2 stimulates the formation of polyubiquitin chains initiating from a single ubiquitin molecule (6 ). In contrast, if a substrate is bound to the E3, ubiquitin may be preferentially discharged to a lysine residue in the substrate. At least part of the preference for transfer to subwww.acschemicalbiolog y.o rg
strate, as opposed to free ubiquitin, comes from the fact that the bound substrate is present at an effective concentration in the millimoles per liter range while free ubiquitin in the cell (or in most in vitro experiments) is present at ~2 orders of magnitude lower effective concentration. Understanding the biochemical basis for E3-accelerated ubiquitin discharge is a central problem in the ubiquitin field, and several questions have emerged: First, how are the appropriate lysine residues in the substrate selected for conjugation. For some substrates, it has been demonstrated that different turnover rates are achieved by ubiquitination of different lysine residues (7 ), so in biological systems, it may be important that one particular lysine out of many serves as the recipient for the first ubiquitin. There is little evidence that E2s for ubiquitin can impart this type of specificity, but structural data suggest a major role for the E3 in orienting the substrate for appropriate conjugation (8, 9 ). Second, once the initial lysine has been selected, polyubiquitination can occur in a highly
processive manner. It is unclear precisely what biochemical features of the system drive processivity nor is it clear how the structural constraints of a growing ubiquitin chain are reconciled with the requirement that the substrate and E2 likely remain physically associated with the E3 for chain elongation even after many ubiquitin mole cules have been attached to a substrate. Mechanism of Ubiquitin Release from the E2. To begin to answer these questions, Petroski and Deshaies (5 ) examined the kinetics of ubiquitin transfer using the proto typical RING E3, SCFCdc4. SCF complexes are composed of the scaffold Cul1, the adaptor protein Skp1, the substrate receptor Cdc4, and the RING protein Rbx1 (2 ) (Figure 2, panel a). The best characterized SCFCdc4 substrate, Sic1 (2 ), binds to Cdc4 in a phosphorylation-dependent manner and is ubiquitinated by the E2 enzyme Cdc34, which interacts with the RING domain of Rbx1 (10, 11 ). Previous work had demonstrated that the conjugation activity of Cdc34 (either on itself as a surrogate substrate or on free ubiquitin) VOL.1 NO.1 • ACS CHEMICAL BIOLO GY
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Figure 2. Model for polyubiquitin chain specificity and processivity. a) The SCFCdc4 ubiquitin ligase is a large multisubunit protein complex which binds the E2 through the RING domain protein Rbx1, substrate (in this example, Sic1) through the F-box protein (in this example, Cdc4), and Cul1 which acts as a scaffold. The S. cerevisiae E2, Cdc34, contains an “acidic loop” located near its E3 binding site and also includes a low affinity noncovalent ubiquitin binding site, the location of which is not known but could be contiguous with the “acidic loop”. b) Initial monoubiquitination of Sic1 is relatively slow, but subsequent rounds of ubiquitin conjugation to the initial ubiquitin are relatively rapid and specific for K48 due to proper orientation of the attacking ubiquitin’s K48 by the Cdc34 “acidic loop”. c) In the Cdc34 “acidic loop” mutant, the formation of polyubiquitin chains is slow and the linkage is nonspecific due to the lack of appropriate positioning of the attacking ubiquitin. d) In the absence of E3, Cdc34 is still able to catalyze the formation of ubiquitin conjugates through attack of the E2~Ub thiol-ester bond by another ubiquitin, but this reaction is much slower than in the presence of E3. In the absence of free ubiquitin (or absence of available lysines in free ubiquitin), the discharge of ubiquitin from the E2 is very slow, relying solely on the hydrolysis of the thiol-ester bond.
is greatly stimulated by Rbx1, a function that was attributed to association of the RING domain with Cdc34 (6, 10, 11 ), and that Cdc34 is specific for the generation of K48‑linked polyubiquitin chains. In order to dissect individual steps, Petroski and Deshaies performed singleturnover assays, initially focusing on ubiquitin discharge from Cdc34 (Figure 2, panel d). Such single-turnover reactions have the ability to reveal changes in rate constants associated with engagement of Cdc34-S~Ub with SCFCdc4 or substrate–SCFCdc4 complexes. As expected, discharge of ubiquitin was enhanced by the SCF complex, independently of substrate, but when a version of ubiquitin lacking lysine residues (UbK0) was used, the rate of discharge was greatly decreased and 22
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was not stimulated by the SCF. This result indicated a role for lysines in ubiquitin in promoting the discharge reaction, but because the Cdc34-S~UbK0 intermediate is relatively stable, it simultaneously set the stage for an examination of how different molecules attack the thiol-ester. Experiments using wild-type and lysine-free Sic1 (Sic1-K0) demonstrated a requirement for lysine residues in the substrate to promote the UbK0 discharge reaction (Figure 2, panel b). Moreover, SCF-driven discharge using a second ubiquitin molecule to form diubiquitin was dependent upon the presence of Lys-48 in ubiquitin, consistent with the specificity of Cdc34 for building K48-linked chains (Figure 2, panel d). The effect of SCF on the discharge rate was due exclusively
to a 40‑fold enhancement in Vmax, as the presence of the SCF had little impact on the Km for ubiquitin in the diubiquitin synthesis reaction (~600 µM). Chain Specificity and Processivity. Detailed analysis of ubiquitination products on Sic1 proteins containing multiple lysine recipients or a single lysine recipient using wild-type versus K0 ubiquitin revealed that the primary reaction products were Sic1 molecules containing a ubiquitin chain, as opposed to multiple monoubiquitination events (5 ). The implication of this result is that attachment of the first ubiquitin mole cule to the substrate is the rate-limiting step in the conjugation process (Figure 2, panel b). To test this, Petroski and Deshaies generated Sic1 containing a single ubiquitin conjugated to lysine 36 (Sic1-Ub1) and w w w. a c s c h e m i ca l biology.org
compared the rate of its polyubiquitination by SCF–Cdc34 with Sic1 lacking ubiquitin. Surprisingly, the rate of ubiquitination was up to 10-fold higher for Sic1-Ub1 than for Sic1, indicating that the rate of ubiquitin chain extension on a substrate already containing the first ubiquitin is faster than the initial conjugation step (Figure 2, panel b). The molecular details of this effect are likely to underlie the processivity of polyubiquitination. One possible explanation is that the presence of the first ubiquitin places the accepting lysine much closer to the Cdc34-S~Ub bound to the RING domain. Indeed, crystallographic data of substrates bound to SCFCdc4 indicate that the position of the bound E2 could be as much as 50 Å away from the substrate lysine, and this gap could be significantly bridged by the first ubiquitin conjugation step (Figure 2, panel a). Alternatively, Petroski and Deshaies surmised, Cdc34 may have an intrinsic affinity for K48 in ubiquitin relative to lysine residues in substrates. The implication of this idea is that Cdc34 would be capable of more rapidly forming a poly ubiquitin chain because of the enhanced molecular complementarities between Cdc34 and its elongating substrate. To test whether there is something special about K48 in recipient ubiquitin, the authors tested whether the K48R ubiquitin mutant is conjugated faster to Sic1-Ub1 than to Sic1 and found that unlike the case with wild-type Ub there was no rate enhancement (5 ). Minimally, this indicated that acceleration of the second conjugation event required K48 in the recipient ubiquitin. Through a further series of experiments, the authors demonstrated that the rate increase seen for K48 ubiquitin chain extension is an intrinsic feature of Cdc34 and can be traced to a feature which, among E2s, is only found in Cdc34 and Ubc7 orthologs. Alignment of more than 30 human E2s for ubiquitin reveals than Cdc34 and Ubc7 orthologs www.acschemicalbiolog y.o rg
all contain a unique insertion near the active site cysteine residue, which contains several acidic residues. The crystal structure of Ubc7 superimposed on UbcH5b, lacking the “acidic loop”, shows that the loop is on the surface of the enzyme and could potentially influence ubiquitin conjugation (Figure 1, panel b). In fact, mutations in the acidic loop of Cdc34 are largely nonfunctional in vivo (12 ). Kinetic analysis of acidic loop mutations in Cdc34 revealed a complex relationship between the presence of this structural feature and the rates and types of conjugates that can form (5 ). First, Cdc34 acidic loop mutants were highly active in transferring lysine-free ubiquitin to Sic1 as well as in promoting polyubiquitin ation of Sic1, indicating that the acidic loop is not crucial to polymerization of ubiquitin per se (Figure 2, panel c). However, analysis of the types of chains formed indicated that the acidic loop mutants of Cdc34 were very inefficient in the formation of K48‑linked ubiquitin chains. In this sense, the acidic loop mutants can be thought of as maintaining intrinsic conjugation activity while losing selectivity in the formation of degradation-competent K48-linked chains (Figure 2, panel c). Models for Processive Ubiquitin Transfer: Orientation versus Allostery. The data presented by Petroski and Deshaies suggest a model wherein processivity in ubiquitin chain formation by Cdc34 reflects an inherent specificity of Cdc34 for utilization of K48 in the recipient ubiquitin in the conjugation reaction (Figure 2, panel b). Presumably, the acidic loop helps to bind ubiquitin and orient K48 in an optimal position for catalysis, an effect that is revealed in Vmax. The effect of the acidic loop in this model is reminiscent of how the heterodimeric E2, Ubc13–Mms2, functions to orient ubiquitin to achieve selective conjugation at K63 (13 ). In this case, a catalytically inactive Mms2 E2 binds the recipient ubiquitin in a manner that orients K63 in the direction of the active
site cysteine of the associated Ubc13‑S~Ub molecule. The location of the acidic loop in Ubc7, and presumably in Cdc34, is located in a structurally distinct position relative to Mms2 in the Ubc13 complex but could nevertheless perform the analogous orientation function for K48 chain extension. Of additional note is that the Mms2 subunit in the Ubc13–Mms2 heterodimer likely acts to decrease Km for the attacking ubiquitin, which is in contrast to the acidic loop of Cdc34 which has little to no effect on Km; however, in both cases these structural features can increase Vmax. An important question concerns the generality of this model. Indeed, the vast majority of E2s, including UbcH4 and UbcH5, which are capable of functioning with the SCF in vitro, lack the acidic loop feature (Figure 1, panel b). Thus, it is unclear precisely how these enzymes achieve processivity and selectivity. UbcH4 and UbcH5 can be activated by several RING E3s in vitro, including the SCF, although in some cases it appears that they are not as processive as Cdc34. Interestingly, Özkan et al. (14 ) have recently investigated the possibility of an allosteric pathway within the E2 UbcH5b, which may provide some insight into the mechanism of ubiquitin discharge during both the initial monoubiquitination of substrate and the proceeding polyubiqutination reactions. In this work, a computational method called statistical coupling analysis (SCA) (15) was used to determine residues in the E2 family of proteins (345 individual sequences) that have similar patterns of variation over evolution and are hence considered to have coevolved. In addition to the expected cluster of “coupled” hydrophobic core residues, there was a second cluster located near the E2 active site. Using a ubiquitin discharge assay, similar to that used by Petroski and Deshaies, Özkan et al. found that mutation of I88 to alanine, among several mutants examined, had the greatest effect VOL.1 NO.1 • ACS CHEMICAL BIOLO GY
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Sequences proximal to the active site of the E2 can govern the specificity and rate of the polyubiquitin chain synthesis on substrates.
on discharge stimulated by two RING E3s (the anaphase promoting complex (APC) subunit Apc11 and the RING domain of CNOT4) (14 ). The authors suggested that I88 functions in an allosteric network that links the RING binding site with the catalytic apparatus of UbcH5b. However, I88 in UbcH5b is located at the point where the acidic loop of Ubc7 emerges from the conserved 310 helix located adjacent to the E2 active site (Figure 1, panel b). Therefore, I88 may not be part of an allosteric network coupling RING binding to the catalytic cysteine but instead may be playing a role analogous to that of the acidic loop in Cdc34–Ubc7, to optimally orient an attacking lysine toward the thiol-ester bond of the E2~Ub. Petroski and Deshaies found that the acidic loop mutants in Cdc34 formed mixed linkage polyubiquitin chains, indicative of a loss of K48 specificity (5 ). If the UbcH5b I88A mutant is analogous to the Cdc34 acidic loop mutants, then two effects would be predicted. First, rates of substrate-independent discharge of the UbcH5b mutant should be slower, both with and without E3 binding. Second, it should be able to transfer the initial ubiquitin to the E3-bound substrate faster than the wild type yet should form random lysine linkages of lower molecular weight than wild-type UbcH5b. From the limited data available, it is clear that the UbcH5b I88A mutant discharges ubiquitin at a slower rate than wild-type UbcH5b in the presence of Apc11 and CNOT4, consistent with the first prediction. Moreover, when UbcH5b I88A is used in conjunction with the full APC complex and its substrate cyclin B, a drastic reduction in polyubiquitin chain length was observed, instead larger amounts of shorter ubiquitin chains are observed as compared with wild type. Although an allosteric mechanism for facilitating ubiquitin discharge from the E2 is still possible, the phenotype described for the UbcH5b I88A mutant is also consistent with a loss in processivity. Although 24
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these data do not definitively show that the I88A mutant is behaving in a manner that is similar to the Cdc34 acidic loop mutants, they are consistent with the idea that sequences proximal to the active site of the E2 can govern the specificity and rate of the polyubiquitin chain synthesis on substrates. To distinguish between the possibilities of a coupled allosteric pathway within the E2 or an orientational role of the 310 helix (including surrounding residues), it will be necessary to investigate the effect of the UbcH5b I88A mutant, as well as similar mutations in other E2s, using the comprehensive battery of assay methods analogous to those employed to study Sic1 but within the context of different E3-substrate pairs. Although the jury is still out on the generality of this mechanism with respect to controlling processivity and specificity in ubiquitination reactions, this work has uncovered a central feature of Cdc34 that facilitates K48 specific chain elongation in the context of the SCF ubiquitin ligase. Acknowledgment: Our work on the ubiquitin pathway is supported by grants from the National Institute of General Medical Sciences and the National Aging Institute to J.W.H. M.S. is the recipient of an American Cancer Society Postdoctoral Fellowship.
7. Petroski, M. D., and Deshaies, R. J. (2003) Context of multiubiquitin chain attachment influences the rate of Sic1 degradation, Mol. Cell. 11, 1435–1444. 8. Wu, G., Xu, G., Schulman, B. A., Jeffrey, P. D., Harper, J. W., and Pavletich, N. P. (2003) Structure of a betaTrCP1-Skp1-beta-catenin complex: destruction motif binding and lysine specificity of the SCF(beta-TrCP1) ubiquitin ligase, Mol. Cell. 11, 1445–1456. 9. Orlicky, S., Tang, X., Willems, A., Tyers, M., and Sicheri, F. (2003) Structural basis for phosphodependent substrate selection and orientation by the SCFCdc4 ubiquitin ligase, Cell 112, 243–256. 10. Skowyra, D., Koepp, D. M., Kamura, T., Conrad, M. N., Conaway, R. C., Conaway, J. W., Elledge, S. J., and Harper, J. W. (1999) Reconstitution of G1 cyclin ubiquitination with complexes containing SCFGrr1 and Rbx1, Science 284, 662–665. 11. Seol, J. H., Feldman, R. M., Zachariae, W., Shevchenko, A., Correll, C. C., Lyapina, S., Chi, Y., Galova, M., Claypool, J., Sandmeyer, S., Nasmyth, K., Shevchenko, A., and Deshaies, R. J. (1999) Cdc53/cullin and the essential Hrt1 RING-H2 subunit of SCF define a ubiquitin ligase module that activates the E2 enzyme Cdc34, Genes Dev. 13, 1614–1626. 12. Pitluk, Z. W., McDonough, M., Sangan, P., and Gonda, D. K. (1995) Novel CDC34 (UBC3) ubiquitinconjugating enzyme mutants obtained by charge-toalanine scanning mutagenesis, Mol. Cell. Biol. 15, 1210–1219. 13. Chan, N. L., and Hill, C. P. (2001) Defining poly ubiquitin chain topology. Nat. Struct. Biol. 8, 650–652. 14. Özkan, E., Yu, H., and Deisenhofer, J. (2005) Mechanistic insight into the allosteric activation of a ubiquitin-conjugating enzyme by RING-type ubiquitin ligases. Proc. Natl. Acad. Sci. U.S.A. 102, 18890–18895. 15. Lockless, S. W., and Ranganathan, R. (1999) Evolutionarily conserved pathways of energetic connectivity in protein families, Science 286, 295–299.
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