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A plethora of ubiquitin ligases determine the intracellular location and fate of numerous proteins in a substrate-specific manner. However, the mechanisms for these functions are incompletely understood. Most ligases have structurally related RING domains that are critical for ligase activity including the recruitment of ubiquitin conjugating enzymes. Here we probe the function of the RING-CH domain of murine γ-herpesvirus-68 ligase mK3 that functions as an immune evasin by targeting major histocompatibility complex (MHC) class I heavy chains for endoplasmic reticulum-associated degradation (ERAD). Interestingly, mK3 mediates ubiquitin conjugation via ester bonds to S or T residues in addition to conventional isopeptide linkages to K residues. To determine the mechanism of non-K ubiquitination of substrates, we introduced into an mK3 background the RING-CH domains of related viral and cellular MARCH (membrane associated RING-CH) ligases. We found that although a conserved W present in all viral RING-CH domains is critical for mK3 function, sequences outside the RING-CH domain determine whether and which non-lysine substrate residues can be ubiquitinated by mK3. Our findings support the model that viral ligases have evolved a highly effective strategy to optimally orient their RING domain with substrate allowing them to ubiquitinate non-K residues.
Ubiquitin (Ub) conjugation of proteins is one of the most pervasive post-translational modifications, and subtle differences in the extent and linkage of the ubiquitination can have profound effects on the intracellular location and ultimate fate of proteins (1–3). However, the basic process by which proteins are ubiquitinated is highly conserved in evolution and involves the catalytic cascade of three classes of proteins. More specifically, the Ub activating enzyme (E1) forms an ATP-dependent thiolester bond between a cysteine residue in its active site and a C-terminal glycine of an Ub subunit. The Ub is then transferred from the E1 to a cysteine residue of an Ub conjugating enzyme (E2). Finally, an E3 Ub ligase (E3) facilitates the transfer of Ub typically to a ε-NH2 group of a lysine residue of the substrate thus making an isopeptide bond (4). The E3 ligases receive considerable attention because of the fact that they are credited with conferring substrate specificity. There are hundreds of mammalian E3 ligases, most of which contain RING domains defined by characteristically spaced conserved cysteine and histidine residues that form a zinc binding cross-brace structure (5). RING-type E3s are non-enzymatic, but are thought to transfer the Ub from a Ub-charged E2 to their substrate by bringing them into close proximity (6). Importantly, the RING domain of the E3 can influence which E2 is recruited among the more than 30 E2s in mammals. However, the details determining which substrate residues are ubiquitinated and the selective roles of the E3, E2 and other host proteins in this process remain largely undefined.
Recently characterized viral E3 ligases that function in immune evasion are providing several insights into molecular interactions of Ub components and their mammalian substrates. More specifically, the mK3 ligase of γHV68 and the highly homologous kK3 and kK5 ligases (also known as MIR1 and MIR2, respectively) of Kaposi's sarcoma-associated herpesvirus (KSHV) have a variant RING domain termed RING-CH (C4HC3) distinguished by the order of their metal binding cysteine and histidine residues. Furthermore all three of these viral ligases have similar membrane topologies with a RING-CH containing N-terminal domain, two transmembrane domains connected by about a 12 amino acid sequence and a variable C-terminal domain. In addition, all three of these viral ligases include major histocompatibility complex (MHC) class I heavy chains (HCs) as substrates (7–9). Strikingly, all three ligases were recently shown to have the unexpected ability to ubiquitinate non-lysine residues as well as lysine residues on their HC substrates (10–12). What is perhaps most intriguing are the differences between these three viral ligases. kK3 was the first ligase reported capable of ubiquitinating cysteine residues of its substrate, using a thiolester bond analogous to the one used by E1 and E2 Ub components (10). More recent studies suggest that kK5 can also ubiquitinate cysteine residues (11). By contrast, mK3 was the first ligase reported to be capable of ubiquitinating serine or threonine residues on its HC substrate via ester bonds (12).
The kK3/kK5 ligases also differ from mK3 in the substrate degradation pathways they induce. Whereas the kK3 and kK5 ligases induce rapid plasma membrane internalization with substrate degradation in the lysosome, mK3 ligase induces endoplasmic reticulum (ER) dislocation to the cytosol with substrate degradation by the proteasome (7,13–16). In addition, kK3 and particularly kK5 target multiple immune regulatory molecules for degradation including HCs, whereas mK3 ligase predominantly targets only HC for degradation (9,16–20). Related to the above differences, mK3 uniquely binds to the transporter associated with antigen processing (TAP), an ATP-dependent peptide pump that transports MHC I ligands from the cytosol into the ER lumen (21). Indeed, TAP has been proposed to function as an adaptor protein for mK3 orienting it such that it only facilitates the ubiquitination of HC (22). By contrast the molecular basis of substrate discrimination by kK3 and kK5 has not been resolved, although sequences in the HC transmembrane domain and cytoplasmic tail have been implicated (23). In any case, defined differences among these highly homologous viral ligases make them particularly efficacious probes for defining the molecular interactions of host proteins required for the ubiquitination of substrate-specific residues, and the biological consequences of this modification.
Subsequent to the discoveries of the viral ligases, cellular proteins containing a RING-CH and putative transmembrane domains were identified. These proteins are typically called MARCH proteins, an acronym for membrane-associated RING-CH proteins. Ten MARCH proteins were discovered in humans with homologs in mouse (24,25). Although still in earlier stages of characterization, it is already known that many cellular MARCH proteins have diverse and important physiological functions. For example, MARCH I and MARCH VIII proteins regulate CD4 T-cell responses by controlling the level of expression of immune molecules MHC class II and B7.2 on antigen presenting cells (24,26–29). By contrast, MARCH IV and MARCH IX proteins potentially regulate CD8 T-cell responses by controlling the level of expression of MHC class I and intercellular adhesion molecule (ICAM) (24). These four MARCH proteins thus regulate expression of immune molecules similar to the viral MARCH proteins, kK3, kK5 and mK3 making it highly likely the virus stole a progenitor MARCH protein from its host and turned it into an immune evasion protein. In striking contrast MARCH V and MARCH XI appear to be tissue-specific ligases controlling mitochondrial morphology or spermatogenesis, respectively (25,30,31). And MARCH II and MARCH III localize to endosomal vesicles and appear to regulate their recycling by controlling the level of expression of epithelial polarity determinants (24,32,33). Although clearly of considerable interest, much is unknown concerning how MARCH proteins specifically identify and ubiquitinate their physiological targets.
A central question to understanding the function of viral and cellular MARCH proteins is what specifically does the RING-CH domain do? For example, is the RING-CH domain required for the ubiquitination of non-lysine resides? Or more specifically, do subtle differences between the RING-CH domains of mK3 and kK3/kK5 ligases determine whether ester versus thiolester bonds are formed with the substrate. Alternatively, do the RING-CH domains of the cellular MARCH protein also have the capability to ubiquitinate non-lysine residues? The answers to these questions provide key insights into the molecular basis of non-lysine ubiquitination with fundamental implication on how E3s interact with E2s to ubiquitinate specific substrate residues.
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Viral E3 ligases provide novel strategies to resolve several outstanding enigmas regarding the precise mechanisms of substrate ubiquitination and its consequences. The reason for this stems from their potency and known substrate specificity, both consequences of their function as immune evasion proteins of the virus. More specifically, the mK3 ligase encoded by murine herpesvirus γHV68 and the highly homologous kK3 and kk5 ligases of KSHV all include MHC class I HCs as substrates that are targeted to Ub-dependent degradation pathways. Indeed, the targeted degradation of HC molecules by these viral ligases allows cells infected by these herpesviruses to persist in their host by escaping detection by immune CD8 T cells. What has also become clear from recent studies is that these viral ligases are highly efficacious molecular probes for degradation pathways. For example, elegant studies of the kK3 ligase have elucidated an Ub-dependent pathway of rapid substrate endocytosis of plasma membrane proteins resulting in their degradation in the lysosome (7,14–16). By contrast, studies of mK3 ligase have elucidated an Ub-dependent pathway of ERAD (13). There is little doubt that these viral ligases exploit physiological pathways as evidenced by their defined interactions with host proteins as well as the fact they have mammalian cellular homologs (the MARCH proteins).
Substrate specificity is a function of the E3 ligase. However, the molecular interactions conferring substrate specificity are in most cases unknown. Although E3 ligases can potentially bind directly to substrate-specific sequence motifs, this is unlikely to be a universal mechanism because most E3 ligases detect multiple substrates that lack an obvious shared sequence or structural motifs. For example, in addition to HC, viral ligase kK5 targets MICA (MHC class I-related chain A), MICB (MHC class I-related chain B), ICAM-1, CD31/PECAM (platelet/endothelial cell adhesion molecule-1), interferon-γ (IFN-γ) receptor 1 as well as VE-cadherin for lysosomal degradation (17–20,49,50). Furthermore, the viral ligase mK3 specifically ubiquitinates the cytoplasmic tails of HC molecules (12). However, the tails and the transmembrane domains of HC molecules are highly polymorphic and share no obvious conserved sequence motifs. Indeed we show here that an HC with a generic (poly G) tail can be ubiquitinated as long as it has K, S or T residues located near the C-terminus. This finding is in striking support of our previously proposed proximity model, whereby mK3 binds TAP that functions as an adaptor protein to confer HC specificity to mK3-mediated ubiquitination (see model in Figure 9). More specifically, this model speculates that when mK3 binds to TAP it orients its RING-CH domain such that it only ubiquitinates HC [although there can be some collateral damage to TAP in certain cells (51)]. Because all HC molecules bind to TAP before peptide acquisition, mK3 can effectively prevent antigen presentation and thereby CD8 T-cell recognition of infected cells. It seems likely to us that this may be a common paradigm and other E3s will similarly use adaptor molecules to orient their RING domains to confer substrate specificity.
Figure 9. Proposed model depicting how mK3 interacts with host proteins to achieve substrate specificity and to allow its RING-CH domain to be in close proximity and optimally oriented to facilitate the polyubiquitination of HC. Previous reports have demonstrated that mK3 forms a complex with TAP and Tpn, primarily mediated through interactions of mK3′s C-terminal domain and TAP (34). This association allows the N-terminal RING domain of mK3 to be in close proximity and properly oriented to recruit its cognate E2s and to facilitate the ubiquitination of the cytoplasmic tail of MHC I HC upon its entry into the PLC, thereby providing specificity independent of substrate sequence. We believe this model provides a viable general strategy for allowing E3s to maintain their requisite substrate specificity while facilitating the ubiquitination of a highly polymorphic substrate such as HC.
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Another remarkable feature of viral ligases is their ability to ubiquitinate non-K residues of their substrates. Whereas kK3 and kK5 can ubiquitinate cysteine residues of HC substrates, mK3 can ubiquitinate S/T residues of HC substrates (10–12). The molecular basis of this non-K ubiquitination is very intriguing as is the question of its physiological relevance. Central to both of these issues is the precise function of the RING-CH domain in substrate ubiquitination. For example, do the viral RING-CH domains possess unique structures that determine their ability to ubiquitinate non-K residues perhaps, or do the RING-CH domains of cellular E3 have the same capacity to ubiquitinate non-K residues? To address these questions, we initially probed the ubiquitination potential of the RING-CH domains of five representative MARCH proteins in the context of mK3 ligase, i.e. RS. Interestingly, all five of the MARCH RING-CH domains in the context of mK3 were capable of ubiquitinating a wt HC tail, but to various degrees and all less than intact mK3. What was particularly striking was an impaired ability to polyubiquitinate beyond Ub2. The overall lack of efficient polyubiquitination by these RING-CH swaps suggests two non-mutually exclusive explanations. One that the structures and functions of the viral versus cellular RING-CH are different and/or that sequences outside the RING are important to support RING activity. Here we present evidence that both of these explanations are correct, thereby suggesting a model for E3/E2 substrate interaction.
Although the viral and cellular ligases with RING-CH domains share the absolutely conserved core C4HC3 zinc coordinating motif, they have sufficient sequence differences to make viral versus cellular RING-CH type ligases cluster into distinct phylogenetic branches (Figure 3B). Indeed an immediately notable difference in the comparisons of the RING-CH sequences is the amino acid in the second position. Although all three viral ligases compared here have a W residue in the second position of their RING-CH domains (position W9 in mK3), the cellular RING-CHs tested mostly have an R residue. The single exception is the RING-CH domain of MARCH V that like the viral homologs has a W. Interestingly, the MARCH V RING-CH in the context of mK3 was the most effective of the MARCH RING-CH swaps at polyubiquitinating HC. As another reflection of its superior polyubiquitination potential, the MARCH V RING-CH swap also displayed the keenest ability to ubiquitinate a single S residue in the HC tail, albeit considerably weaker than wt mK3 ligase (Figure 4B).
Importantly, a recent solution structure showed the W residue in the second position of the RING-CH domain of viral ligase kK3 forms a novel hydrophobic pocket with a second W residue between the sixth and seventh zinc coordinating residues that is highly conserved among all viral and cellular RING-type E3s (shown in Figure 8A) (47). It should be noted that this highly conserved W residue between the sixth and seventh zinc coordinating residues is involved in E2 recruitment as shown in previously published studies of c-Cbl and kK3 (14,52). To define the functional importance of W in the second RING-CH position of viral ligases and its possible importance for non-K ubiquitinations, we mutated the W to R in mK3. This mutant, mK3 W9R, resulted in a dramatic kinetic impairment of Ub chain assembly compared to wt mK3 ligase. Remarkably, this kinetic impairment rendered the mK3 W9R mutant functionally inert in that it was no longer capable of destabilizing HC as shown in a pulse/chase comparison with wt mK3 (Figure 7B). Based on the model shown in Figure 8B, the W9R mutation of mK3 is predicted to drastically reconfigure the topology of the aforementioned hydrophobic pocket implicated in E2 interaction of other E3 ligases. Thus, it seems likely that the mK3 W9R mutant interacts poorly with its cognate E2 or associates with a different E2 than wt mK3, possibly resulting in aberrant Ub chain assembly. Alternatively, mK3 may recruit two E2 conjugases similar to kK3 which recruits UbcH5b/c and Ubc13 that are required for targeting HC degradation in the lysosome (48). However, in the case of mK3 we would speculate that the first E2 might transfer a premade Ub2 chain to HC and the second E2 might facilitate the further polyubiquitination needed to induce ERAD. If this model is correct, the W9R mutation may have affected only interaction with the second E2. In any case, we show here for the first time the W residue in the second RING-CH position (between the first and second zinc coordinating residues) is critical for mK3 function, but it is not a critical factor determining the ability to ubiquitinate non-K residues.
Consistent with the similarity among viral RING-CH domains, chimeric constructs with the kK3 or kK5 RING-CH domains in the context of mK3 were clearly capable of robustly polyubiquitinating HC substrates via K, S or T residues (Figure 5). This finding was of considerable interest because intact kK3 and kK5 can efficiently only ubiquitinate K or C residues, but not S/T residues. Another interesting result was that in contrast to polyubiquitination of HC by intact kK3 and kK5 (14,53), Ub2 is the minimal form of ubiquitinated HC observed in the presence of the kK3 and kK5 RS. In addition, we also found that the kK3 RS and kK5 RS have the same target residue positional preferences as wt mK3. Thus, our findings demonstrate unequivocally that the RING-CH domain does not function autonomously in its ability to make ester versus thiolester Ub linkages, conferring substrate residue positional constraints, or in determining the mechanism of Ub chain assembly on substrates. In further support of these conclusions, we show here that the DIRT is incredibly important for mK3 ligase activity. More specifically, we found that in the context of mK3 the DIRT from kK3 but not kK5 supported ubiquitination of HC substrates. Consistent with its ability to support ubiquitination, the sequence of the kK3 DIRT is considerably more similar to that of mK3 than it is to the sequence of the kK5 DIRT. Thus consistent with recent studies of other E3 ligases, the RING domain of mK3 does not function autonomously to ubiquitinate K or S/T residues of HC substrates (24,54–57).
Cumulative findings reported here suggest a model by which mK3 ubiquitinates substrates, with implications on its ability to form ester bonds. Clearly the RING-CH domain itself does not determine the ability to form ester versus thiolester bonds. The importance of the DIRT between the RING-CH and transmembrane (TM) is also of critical importance and could be directly involved in E2 binding as published with other ligases. Alternatively, the DIRT could optimally orient the RING domain for substrate interaction. Indeed an orientation model was recently proposed for the interaction of the kK3 and kK5 viral ligases with HC whereby their respective DIRTs differentially interact with the membrane proximal sequences in their C-terminal domains (11). It was further speculated that the differential juxtaposition of these two membrane proximal sequences in kK3 versus kK5 differentially affects the location of residues on the HC tail that were preferentially ubiquitinated by kK3 versus kK5. This model has similarities to our proximity model, whereby TAP binding with the C-terminal domain of mK3 orients the RING-CH domain and thereby impacts on the location of optimally ubiquitinated residues. It should also be noted that membrane proximal regions of the C-terminal domain of kK3 and kK5 contain putative protein-interaction domains, suggesting they might also interact with adapter proteins that could also influence substrate specificity (16). In any case, it is the location on the tail of the ubiquitinated residue (S/T or K) and not surrounding sequences that is important for mK3 activity, thus highly accentuating the importance of RING orientation/proximity to its substrate. Based on these combined findings, we propose that what is unique about mK3 and perhaps other viral ligases is their optimal positioning of the RING-CH domain as rendered by its interaction with the DIRT and host adaptor proteins. It is attractive to speculate this putative optimized activity of the viral ligase is reflective of its divergence from host ligases to more proficiently ubiquitinate substrates and thereby function as an immune evasin. If this model is correct, a specific E2 uniquely capable of forming ester or thiolester bonds need not be recruited. In support of this conclusion, in comparisons of the numerous mutant and chimeric mK3 constructs we have tested, the ones found most active at ubiquitinating wt HC were also capable of ubiquitinating HC constructs with optimally placed S, T or K tail residues. Thus, we propose that the data is best explained by viral ligases having evolved a strategy for establishing an optimal topological orientation of their RING-CH domains with substrate thereby allowing the ubiquitination of S/T or K residues.
The selective pressures on viral RING-CH E3 ligases have favored greater target residue flexibility compared to most cellular ligases; however, it should be noted that there is increasing evidence for the physiological importance of substrate ubiquitination via thiolester/ester bonds (e.g. TCRa, CD4, Bid) (58–60). Most notably, in yeast and mammals export of Pex5p, the peroxisomal import receptor, requires ubiquitination of a conserved C residue of Pex5p (61–63). Interestingly, this thiolester bond formation in mammals appears to be mediated by members of the UBE2D family that are thought to be responsible for a large fraction of the total ubiquitinated proteins (64,65). Thus, these findings are highly complementary to our findings in suggesting general applications of non-K ubiquitination and the fact that they need not be mediated by specialized E2s.