A single MIU motif of MINDY‐1 recognizes K48‐linked polyubiquitin chains

The eight different types of ubiquitin (Ub) chains that can be formed play important roles in diverse cellular processes. Linkage‐selective recognition of Ub chains by Ub‐binding domain (UBD)‐containing proteins is central to coupling different Ub signals to specific cellular responses. The motif interacting with ubiquitin (MIU) is a small UBD that has been characterized for its binding to monoUb. The recently discovered deubiquitinase MINDY‐1/FAM63A contains a tandem MIU repeat (tMIU) that is highly selective at binding to K48‐linked polyUb. We here identify that this linkage‐selective binding is mediated by a single MIU motif (MIU2) in MINDY‐1. The crystal structure of MIU2 in complex with K48‐linked polyubiquitin chains reveals that MIU2 on its own binds to all three Ub moieties in an open conformation that can only be accommodated by K48‐linked triUb. The weak Ub binder MIU1 increases overall affinity of the tMIU for polyUb chains without affecting its linkage selectivity. Our analyses reveal new concepts for linkage selectivity and polyUb recognition by UBDs.     

Mechanistic insights into enhancement or inhibition of phase separation by different polyubiquitin chains

Ubiquitin‐binding shuttle UBQLN2 mediates crosstalk between proteasomal degradation and autophagy, likely via interactions with K48‐ and K63‐linked polyubiquitin chains, respectively. UBQLN2 comprises self‐associating regions that drive its homotypic liquid–liquid phase separation (LLPS). Specific interactions between one of these regions and ubiquitin inhibit UBQLN2 LLPS. Here, we show that, unlike ubiquitin, the effects of multivalent polyubiquitin chains on UBQLN2 LLPS are highly dependent on chain types. Specifically, K11‐Ub4 and K48‐Ub4 chains generally inhibit UBQLN2 LLPS, whereas K63‐Ub4, M1‐Ub4 chains, and a designed tetrameric ubiquitin construct significantly enhance LLPS. We demonstrate that these opposing effects stem from differences in chain conformations but not in affinities between chains and UBQLN2. Chains with extended conformations and increased accessibility to the ubiquitin‐binding surface promote UBQLN2 LLPS by enabling a switch between homotypic to partially heterotypic LLPS that is driven by both UBQLN2 self‐interactions and interactions between multiple UBQLN2 units with each polyubiquitin chain. Our study provides mechanistic insights into how the structural and conformational properties of polyubiquitin chains contribute to heterotypic LLPS with ubiquitin‐binding shuttles and adaptors.     

Improved quantitative mass spectrometry methods for characterizing complex ubiquitin signals

Ubiquitinated substrates can be recruited to macromolecular complexes through interactions between their covalently bound ubiquitin (Ub) signals and Ub receptor proteins. To develop a functional understanding of the Ub system in vivo, methods are needed to determine the composition of Ub signals on individual substrates and in protein mixtures. Mass spectrometry has emerged as an important tool for characterizing the various forms of Ub. In the Ubiquitin-AQUA approach, synthetic isotopically labeled internal standard peptides are used to quantify unbranched peptides and the branched -GG signature peptides generated by trypsin digestion of Ub signals. Here we have built upon existing methods and established a comprehensive platform for the characterization of Ub signals. Digested peptides and isotopically labeled standards are analyzed either by selected reaction monitoring on a QTRAP mass spectrometer or by narrow window extracted ion chromatograms on a high resolution LTQ-Orbitrap. Additional peptides are now monitored to account for the N terminus of ubiquitin, linear polyUb chains, the peptides surrounding K33 and K48, and incomplete digestion products. Using this expanded battery of peptides, the total amount of Ub in a sample can be determined from multiple loci within the protein, minimizing possible confounding effects of complex Ub signals, digestion abnormalities, or use of mutant Ub in experiments. These methods have been useful for the characterization of in vitro, multistage ubiquitination and have now been extended to reactions catalyzed by multiple E2 enzymes. One question arising from in vitro studies is whether individual protein substrates in cells may be modified by multiple forms of polyUb. Here we have taken advantage of recently developed polyubiquitin linkage-specific antibodies recognizing K48- and K63-linked polyUb chains, coupled with these mass spectrometry methods, to further evaluate the abundance of mixed linkage Ub substrates in cultured mammalian cells. By combining these two powerful tools, we show that polyubiquitinated substrates purified from cells can be modified by mixtures of K48, K63, and K11 linkages.     

Structural and biochemical studies of the open state of Lys48-linked diubiquitin

Ubiquitin (Ub) is a small protein highly conserved among eukaryotes and involved in practically all aspects of eukaryotic cell biology. Polymeric chains assembled from covalently-linked Ub monomers function as molecular signals in the regulation of a host of cellular processes. Our previous studies have shown that the predominant state of Lys48-linked di- and tetra-Ub chains at near-physiological conditions is a closed conformation, in which the Ub-Ub interface is formed by the hydrophobic surface residues of the adjacent Ub units. Because these very residues are involved in (poly)Ub interactions with the majority of Ub-binding proteins, their sequestration at the Ub-Ub interface renders the closed conformation of polyUb binding incompetent. Thus the existence of open conformation(s) and the interdomain motions opening and closing the Ub-Ub interface is critical for the recognition of Lys48-linked polyUb by its receptors. Knowledge of the conformational properties of a polyUb signal is essential for our understanding of its specific recognition by various Ub-receptors. Despite their functional importance, open states of Lys48-linked chains are poorly characterized. Here we report a crystal structure of the open state of Lys48-linked di-Ub. Moreover, using NMR, we examined interactions of the open state of this chain (at pH4.5) with a Lys48-linkage-selective receptor, the UBA2 domain of a shuttle protein hHR23a. Our results show that di-Ub binds UBA2 in the same mode and with comparable affinity as the closed state. Our data suggest a mechanism for polyUb signal recognition, whereby Ub-binding proteins select specific conformations out of the available ensemble of polyUb chain conformations. This article is part of a Special Issue entitled: Ubiquitin Drug Discovery and Diagnostics.     

Conformational dynamics of wild-type Lys-48-linked diubiquitin in solution

Proteasomal degradation is mediated through modification of target proteins by Lys-48-linked polyubiquitin (polyUb) chain, which interacts with several binding partners in this pathway through hydrophobic surfaces on individual Ub units. However, the previously reported crystal structures of Lys-48-linked diUb exhibit a closed conformation with sequestered hydrophobic surfaces. NMR studies on mutated Lys-48-linked diUb indicated a pH-dependent conformational equilibrium between closed and open states with the predominance of the former under neutral conditions (90% at pH 6.8). To address the question of how Ub-binding proteins can efficiently access the sequestered hydrophobic surfaces of Ub chains, we revisited the conformational dynamics of Lys-48-linked diUb in solution using wild-type diUb and cyclic forms of diUb in which the Ub units are connected through two Lys-48-mediated isopeptide bonds. Our newly determined crystal structure of wild-type diUb showed an open conformation, whereas NMR analyses of cyclic Lys-48-linked diUb in solution revealed that its structure resembled the closed conformation observed in previous crystal structures. Comparison of a chemical shift of wild-type diUb with that of monomeric Ub and cyclic diUb, which mimic the open and closed states, respectively, with regard to the exposure of hydrophobic surfaces to the solvent indicates that wild-type Lys-48-linked diUb in solution predominantly exhibits the open conformation (75% at pH 7.0), which becomes more populated upon lowering pH. The intrinsic properties of Lys-48-linked Ub chains to adopt the open conformation may be advantageous for interacting with Ub-binding proteins.     

A SnapShot of Ubiquitin Chain Elongation: LYSINE 48-TETRA-UBIQUITIN SLOWS DOWN UBIQUITINATION*

We have explored the mechanisms of polyubiquitin chain assembly with reconstituted ubiquitination of IκBα and β-catenin by the Skp1-cullin 1-βTrCP F-box protein (SCF^βTrCP) E3 ubiquitin (Ub) ligase complex. Competition experiments revealed that SCF^βTrCP formed a complex with IκBα and that the Nedd8 modified E3-substrate platform engaged in dynamic interactions with the Cdc34 E2 Ub conjugating enzyme for chain elongation. Using “elongation intermediates” containing β-catenin linked with Ub chains of defined length, it was observed that a Lys-48-Ub chain of a length greater than four, but not its Lys-63 linkage counterparts, slowed the rate of additional Ub conjugation. Thus, the Ub chain length and linkage impact kinetic rates of chain elongation. Given that Lys-48-tetra-Ub is packed into compact conformations due to extensive intrachain interactions between Ub subunits, this topology may limit the accessibility of SCF^βTrCP/Cdc34 to the distal Ub Lys-48 and result in slowed elongation.     

Exploring the Linkage Dependence of Polyubiquitin Conformations Using Molecular Modeling

Posttranslational modification of proteins by covalent attachment of a small protein ubiquitin (Ub) or a polymeric chain of Ub molecules (called polyubiquitin) is involved in controlling a vast variety of processes in eukaryotic cells. The question of how different polyubiquitin signals are recognized is central to understanding the specificity of various types of polyubiquitination. In polyubiquitin, monomers are linked to each other via an isopeptide bond between the C-terminal glycine of one Ub and a lysine of the other. The functional outcome of polyubiquitination depends on the particular lysine involved in chain formation and appears to rely on linkage-dependent conformation of polyubiquitin. Thus, K48-linked chains, a universal signal for proteasomal degradation, under physiological conditions adopt a closed conformation where functionally important residues L8, I44, and V70 are sequestered at the interface between two adjacent Ub monomers. By contrast, K63-linked chains, which act as a nonproteolytic regulatory signal, adopt an extended conformation that lacks hydrophobic interubiquitin contact. Little is known about the functional roles of the so-called “noncanonical” chains (linked via K6, K11, K27, K29, or K33, or linked head-to-tail), and no structural information on these chains is available, except for information on the crystal structure of the head-to-tail-linked diubiquitin (Ub₂). In this study, we use molecular modeling to examine whether any of the noncanonical chains can adopt a closed conformation similar to that in K48-linked polyubiquitin. Our results show that the eight possible Ub₂ chains can be divided into two groups: chains linked via K6, K11, K27, or K48 are predicted to form a closed conformation, whereas chains linked via K29, K33, or K63, or linked head-to-tail are unable to form such a contact due to steric occlusion. These predictions are validated by the known structures of K48-, K63-, and head-to-tail-linked chains. Our study also predicts structural models for Ub₂ chains linked via K6, K11, or K27. The implications of these findings for linkage-selective recognition of noncanonical polyubiquitin signals by various receptors are discussed.     

Molecular determinants of polyubiquitin linkage selection by an HECT ubiquitin ligase

Ubiquitin (Ub)-protein ligases (E3s) frequently modify their substrates with multiple Ub molecules in the form of a polyubiquitin (poly-Ub) chain. Although structurally distinct poly-Ub chains (linked through different Ub lysine (Lys) residues) can confer different fates on target proteins, little is known about how E3s select the Lys residue to be used in chain synthesis. Here, we used a combination of mutagenesis, biochemistry, and mass spectrometry to map determinants of linkage choice in chain assembly catalyzed by KIAA10, an HECT (Homologous to E6AP C-Terminus) domain E3 that synthesizes K29- and K48-linked chains. Focusing on the Ub molecule that contributes the Lys residue for chain formation, we found that specific surface residues adjacent to K48 and K29 are critical for the usage of the respective Lys residues in chain synthesis. This direct mechanism of linkage choice bears similarities to the mechanism of substrate site selection in sumoylation catalyzed by Ubc9, but is distinct from the mechanism of chain linkage selection used by the Mms2/Ubc13 (Ub E2 variant (UEV)/E2) complex.     

Different HECT domain ubiquitin ligases employ distinct mechanisms of polyubiquitin chain synthesis

Individual ubiquitin (Ub)-protein ligases (E3s) cooperate with specific Ub-conjugating enzymes (E2s) to modify cognate substrates with polyubiquitin chains. E3s belonging to the Really Interesting New Gene (RING) and Homologous to E6-Associated Protein (E6AP) C-Terminus (HECT) domain families utilize distinct molecular mechanisms. In particular, HECT E3s, but not RING E3s, form a thiol ester with Ub before transferring Ub to the substrate lysine. Here we report that different HECT domain E3s can employ distinct mechanisms of polyubiquitin chain synthesis. We show that E6AP builds up a K48-linked chain on its HECT cysteine residue, while KIAA10 builds up K48- and K29-linked chains as free entities. A small region near the N-terminus of the conserved HECT domain helps to bring about this functional distinction. Thus, a given HECT domain can specify both the linkage of a polyubiquitin chain and the mechanism of its assembly.     

Modulation of K11-linkage formation by variable loop residues within UbcH5A

Ubiquitination refers to the covalent addition of ubiquitin (Ub) to substrate proteins or other Ub molecules via the sequential action of three enzymes (E1, E2, and E3). Recent advances in mass spectrometry proteomics have made it possible to identify and quantify Ub linkages in biochemical and cellular systems. We used these tools to probe the mechanisms controlling linkage specificity for UbcH5A. UbcH5A is a promiscuous E2 enzyme with an innate preference for forming polyubiquitin chains through lysine 11 (K11), lysine 48 (K48), and lysine 63 (K63) of Ub. We present the crystal structure of a noncovalent complex between Ub and UbcH5A. This structure reveals an interaction between the Ub surface flanking K11 and residues adjacent to the E2 catalytic cysteine and suggests a possible role for this surface in formation of K11 linkages. Structure-guided mutagenesis, in vitro ubiquitination and quantitative mass spectrometry have been used to characterize the ability of residues in the vicinity of the E2 active site to direct synthesis of K11- and K63-linked polyubiquitin. Mutation of critical residues in the interface modulated the linkage specificity of UbcH5A, resulting in generation of more K63-linked chains at the expense of K11-linkage synthesis. This study provides direct evidence that the linkage specificity of E2 enzymes may be altered through active-site mutagenesis.     

Crystal structure of cyclic Lys48-linked tetraubiquitin

Lys48-linked polyubiquitin chains serve as a signal for protein degradation by 26S proteasomes through its Ile44 hydrophobic patches interactions. The individual ubiquitin units of each chain are conjugated through an isopeptide bond between Lys48 and the C-terminal Gly76 of the preceding units. The conformation of Lys48-linked tetraubiquitin has been shown to change dynamically depending on solution pH. Here we enzymatically synthesized a wild-type Lys48-linked tetraubiquitin for structural study. In the synthesis, cyclic and non-cyclic species were obtained as major and minor fractions, respectively. This enabled us to solve the crystal structure of tetraubiquitin exclusively with native Lys48-linkages at 1.85 Å resolution in low pH 4.6. The crystallographic data clearly showed that the C-terminus of the first ubiquitin is conjugated to the Lys48 residue of the fourth ubiquitin. The overall structure is quite similar to the closed form of engineered tetraubiquitin at near-neutral pH 6.7, previously reported, in which the Ile44 hydrophobic patches face each other. The structure of the second and the third ubiquitin units [Ub(2)-Ub(3)] connected through a native isopeptide bond is significantly different from the conformations of the corresponding linkage of the engineered tetraubiquitins, whereas the structures of Ub(1)-Ub(2) and Ub(3)-Ub(4) isopeptide bonds are almost identical to those of the previously reported structures. From these observations, we suggest that the flexible nature of the isopeptide linkage thus observed contributes to the structural arrangements of ubiquitin chains exemplified by the pH-dependent closed-to-open conformational transition of tetraubiquitin.     

Mammalian Ddi2 is a shuttling factor containing a retroviral protease domain that influences binding of ubiquitylated proteins and proteasomal degradation

Although several proteasome subunits have been shown to bind ubiquitin (Ub) chains, many ubiquitylated substrates also associate with 26S proteasomes via “shuttling factors.” Unlike the well-studied yeast shuttling factors Rad23 and Dsk2, vertebrate homologs Ddi2 and Ddi1 lack a Ub-associated domain; therefore, it is unclear how they bind Ub. Here, we show that deletion of Ddi2 leads to the accumulation of Ub conjugates with K11/K48 branched chains. We found using affinity copurifications that Ddi2 binds Ub conjugates through its Ub-like domain, which is also required for Ddi2 binding to proteasomes. Furthermore, in cell extracts, adding Ub conjugates increased the amount of Ddi2 associated with proteasomes, and adding Ddi2 increased the binding of Ub conjugates to purified proteasomes. In addition, Ddi2 also contains a retroviral protease domain with undefined cellular roles. We show that blocking the endoprotease activity of Ddi2 either genetically or with the HIV protease inhibitor nelfinavir increased its binding to Ub conjugates but decreased its binding to proteasomes and reduced subsequent protein degradation by proteasomes leading to further accumulation of Ub conjugates. Finally, nelfinavir treatment required Ddi2 to induce the unfolded protein response. Thus, Ddi2 appears to function as a shuttling factor in endoplasmic reticulum–associated protein degradation and delivers K11/K48-ubiquitylated proteins to the proteasome. We conclude that the protease activity of Ddi2 influences this shuttling factor activity, promotes protein turnover, and helps prevent endoplasmic reticulum stress, which may explain nelfinavir’s ability to enhance cell killing by proteasome inhibitors.     

Cyclization of polyubiquitin by the E2-25K ubiquitin conjugating enzyme

For most substrates of ubiquitin (Ub)-dependent degradation, recognition by the proteasome is mediated by a covalently attached signal assembled from multiple ubiquitins linked to each other via the C terminus of one Ub and the epsilon-amine of Lys(48) of another Ub. Among Ub-conjugating enzymes, E2-25K is unique in its ability to synthesize in vitro unanchored Lys(48)-linked poly-Ub chains from mono- or poly-Ub, E1, and ATP; thus, E2-25K has distinct binding sites for donor and acceptor (poly)Ub. During studies of chain assembly by E2-25K, we observed that Lys(48)-linked tri-Ub was efficiently converted to a new species that upon SDS-polyacrylamide gel electrophoresis migrated between linear di-Ub and tri-Ub. Analysis of this product by mass spectrometry and tryptic digestion showed that it was a cyclic form of tri-Ub. Cyclization of tri-Ub requires E1, E2-25K, ATP, and that the linear substrate has a free Gly(76) C terminus on the proximal end Ub and a Lys(48) side chain available on the distal end Ub. E2-25K similarly can catalyze the cyclization of longer poly-Ub chains, including tetra- and penta-Ub. Although cyclic tri-Ub resists hydrolysis by the PA700 or isopeptidase T deubiquitinating enzymes, it can be disassembled to Ub monomers by isopeptidase(s) in a red blood cell extract. Thus, if cyclic poly-Ub forms in vivo, it will not accumulate as a dead-end product.     

A 25-kilodalton ubiquitin carrier protein (E2) catalyzes multi-ubiquitin chain synthesis via lysine 48 of ubiquitin

Target protein multi-ubiquitination involving lysine 48 of ubiquitin (Ub) is known to occur during protein degradation in the ATP- and Ub-dependent proteolytic pathway (Chau, V., Tobias, J. W., Bachmair, A., Marriott, D., Ecker, D. J., Gonda, D. K., and Varshavsky, A. (1989) Science 243, 1576-1583). However, little is known about the enzymatic mechanism of multi-ubiquitination. We show that a purified Ub carrier protein, E2(25)K, catalyzes multi-Ub chain synthesis from purified Ub. Incubation of E2(25)K with Ub activating enzyme (E1), MgATP, and radiolabeled Ub (Mr = 8500) resulted in time dependent appearance of a "ladder" of radiolabeled Ub conjugates with molecular masses of 8.5n kDa, where n = 1, 2, 3, 4... (up to at least n = 10). The kinetics of this conjugative process were consistent with Ub2 acting as a steady-state intermediate. The putative Ub2 product of E2(25)K catalysis was purified and cleaved with a partially purified isopeptidase preparation. The sole cleavage product (Mr = 8500) had a tryptic digest identical to that of authentic Ub, confirming that the original conjugate was Ub2. Tryptic digestion of intact Ub2 gave products consistent with the existence of an isopeptide linkage between the COOH terminus of one Ub and Lys-48 of the other; this structure was confirmed by sequence analysis of the unique Ub2 tryptic fragment. Tryptic digestion of higher order Ubn adducts (n greater than or equal to 4) yielded fragments identical to those of Ub2, indicating that E2(25)K ligates successive Ub molecules primarily or exclusively via Lys-48. Although several other E2s supported synthesis of an apparent Ub2 adduct of undetermined linkage, only E2(25)K was capable of synthesizing multi-Ub chains from isolated Ub. Quantitative analysis of single turnovers showed that transfer from E2(25)K-Ub to Ub and Ub2 occurred with kappa 2 = 488 and 1170 M-1 min-1, respectively, at pH 7.3 and 37 degrees C. These results show that increasing the number of Ub molecules in a chain increases susceptibility to further ubiquitination by E2(25)K. Ub2 was a good substrate for activation by E1 and was readily transferred to E2(25)K. The labile E2(25)K-Ub2 adduct was catalytically active, and exhibited preference for Ub2 (versus Ub) as acceptor. These results suggest that E2(25)K may function as a multi-ubiquitinating enzyme in the Ub-dependent proteolytic pathway.     

Conformational transition states of a beta-hairpin peptide between the ordered and disordered conformations in explicit water

The conformational transition states of a beta-hairpin peptide in explicit water were identified from the free energy landscapes obtained from the multicanonical ensemble, using an enhanced conformational sampling calculation. The beta-hairpin conformations were significant at 300 K in the landscape, and the typical nuclear Overhauser effect signals were reproduced, consistent with the previously reported experiment. In contrast, the disordered conformations were predominant at higher temperatures. Among the stable conformations at 300 K, there were several free energy barriers, which were not visible in the landscapes formed with the conventional parameters. We identified the transition states around the saddle points along the putative folding and unfolding paths between the beta-hairpin and the disordered conformations in the landscape. The characteristic features of these transition states are the predominant hydrophobic contacts and the several hydrogen bonds among the side-chains, as well as some of the backbone hydrogen bonds. The unfolding simulations at high temperatures, 400 K and 500 K, and their principal component analyses also provided estimates for the transition state conformations, which agreed well with those at 400 K and 500 K deduced from the current free energy landscapes at 400 K and 500 K, respectively. However, the transition states at high temperatures were much more widely distributed on the landscape than those at 300 K, and their conformations were different.     

A perturbed ubiquitin landscape distinguishes between ubiquitin in trafficking and in proteolysis

Any of seven lysine residues on ubiquitin can serve as the base for chain-extension, resulting in a sizeable spectrum of ubiquitin modifications differing in chain length or linkage type. By optimizing a procedure for rapid lysis, we charted the profile of conjugated cellular ubiquitin directly from whole cell extract. Roughly half of conjugated ubiquitin (even at high molecular weights) was nonextended, consisting of monoubiquitin modifications and chain terminators (endcaps). Of extended ubiquitin, the primary linkages were via Lys48 and Lys63. All other linkages were detected, contributing a relatively small portion that increased at lower molecular weights. In vivo expression of lysineless ubiquitin (K0 Ub) perturbed the ubiquitin landscape leading to elevated levels of conjugated ubiquitin, with a higher mono-to-poly ratio. Affinity purification of these trapped conjugates identified a comprehensive list of close to 900 proteins including novel targets. Many of the proteins enriched by K0 ubiquitination were membrane-associated, or involved in cellular trafficking. Prime among them are components of the ESCRT machinery and adaptors of the Rsp5 E3 ubiquitin ligase. Ubiquitin chains associated with these substrates were enriched for Lys63 linkages over Lys48, indicating that K0 Ub is unevenly distributed throughout the ubiquitinome. Biological assays validated the interference of K0 Ub with protein trafficking and MVB sorting, minimally affecting Lys48-dependent turnover of proteasome substrates. We conclude that despite the shared use of the ubiquitin molecule, the two branches of the ubiquitin machinery--the ubiquitin-proteasome system and the ubiquitin trafficking system--were unevenly perturbed by expression of K0 ubiquitin.     

Molecular Recognition of M1-Linked Ubiquitin Chains by Native and Phosphorylated UBAN Domains

Although the Ub-binding domain in ABIN proteins and NEMO (UBAN) is highly conserved, UBAN-containing proteins exhibit different Ub-binding properties, resulting in their diverse biological roles. Post-translational modifications further control UBAN domain specificity for poly-Ub chains. However, precisely, how the UBAN domain structurally confers such functional diversity remains poorly understood. Here we report crystal structures of ABIN-1 alone and in complex with one or two M1-linked di-Ub chains. ABIN-1 UBAN forms a homo-dimer that provides two symmetrical Ub-binding sites on either side of the coiled-coil structure. Moreover, crystal structures of ABIN1 UBAN in complex with di-Ub chains reveal a concentration-dependency of UBAN/di-Ub binding stoichiometry. Analysis of UBAN/M1-linked di-Ub binding characteristics indicates that phosphorylated S473 in OPTN and its corresponding phospho-mimetic residue in ABIN-1 (E484) are essential for high affinity interactions with M1-linked Ub chains. Also, a phospho-mimetic mutation of A303 in NEMO, corresponding to S473 of OPTN, increases binding affinity for M1-linked Ub chains. These findings are in line with the diverse physiological roles of UBAN domains, as phosphorylation of OPTN UBAN is required to enhance its binding to Ub during mitophagy.     

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.