Acceptor Subsite Mutants of Limosilactobacillus fermentum NCC 2970 GtfB 4,3-α-Glucanotransferase Regulate the Ratio of (α1 → 3)/(α1 → 6) Linkages in Biosynthesized α-Glucans.

Limosilactobacillus fermentum NCC 2970 GtfB (Lf2970 GtfB) is the only characterized 4,3-α-glucanotransferase (4,3-α-GTase) in the glycoside hydrolase (GH) 70 family belonging to the GtfB subfamily. However, the mechanism for its (α1 → 3) linkage formation remains unclear, and the structural determinants of its linkage specificity remain to be explored. Here, sequence alignment and structural comparison were conducted to identify key amino acids that may be critical for linkage specificity. Five residues of Lf2970 GtfB (D991, G1028, A1398, T1400, and E1405), located at donor and acceptor subsites, were selected for mutation. Product structure analysis revealed that D991 and G1028, located near the acceptor binding subsites, played crucial roles in linkage formation. Besides native (α1 → 4) and (α1 → 3) linkages, mutants G1028R and D991N showed 8 and 10% (α1 → 6) linkage increases compared to 1% for wild-type in products. Additionally, molecular docking studies demonstrated that the orientation of acceptor binding in G1028R and D991N mutants was favorable for (α1 → 6) linkage synthesis. However, the mutation at positions A1398, T1400, and E1405 indicated that the donor subsites contribute less to the linkage specificity. These results shed light on the structural determinants of linkage specificity of 4,3-α-GTase Lf2970 GtfB and provided insights into the structure-function relationship of family GH70.

The Ufd1 cofactor determines the linkage specificity of polyubiquitin chain engagement by the AAA+ ATPase Cdc48.

The AAA+ ATPase Cdc48 utilizes the cofactor Ufd1/Npl4 to bind and thread polyubiquitinated substrates for their extraction from complexes or membranes and often for subsequent proteasomal degradation. Previous studies indicated that Cdc48 engages polyubiquitin chains through the Npl4-mediated unfolding of an initiator ubiquitin; yet, the underlying principles remain largely unknown. Using FRET-based assays, we revealed the mechanisms and kinetics of ubiquitin unfolding, insertion into the ATPase, and unfolding of the ubiquitin-attached substrate. We found that Cdc48 uses Ufd1's UT3 domain to bind a K48-linked ubiquitin on the initiator's proximal side of the chain, thereby directing the initiator toward rapid unfolding by Npl4 and engagement by Cdc48. Ubiquitins on the initiator's distal side increase substrate affinity and facilitate unfolding but impede substrate release from Cdc48-Ufd1/Npl4 in the absence of additional cofactors. Our findings explain how Cdc48-UN efficiently processes substrates with K48-linked chains of 4-6 ubiquitins, which represent most cellular polyubiquitinated proteins.

Mutations in the Different Residues between Dextransucrase Gtf-DSM and Reuteransucrase GtfO for the Investigation of Linkage Specificity Determinants.

The dextransucrase Gtf-DSM has 99.3% sequence identity with the reuteransucrase GtfO, and only 11 out of 1045 residues are different between their N-terminally truncated recombinant forms. Gtf-DSM is capable of synthesizing a dextran with 1% (α1 → 2), 6% (α1 → 4), 24% (α1 → 3), and 69% (α1 → 6) linkages, while GtfO produces a reuteran with 21% (α1 → 6) and 79% (α1 → 4) linkages. In this work, using recombinant Gtf-DSM and GtfO as templates, parallel substitutions targeting these 11 distinguishing residues were performed to investigate their linkage specificity determinants. The combinatorial mutation (I937L/D977A/D1083V/Q1086K/K1087G) at the acceptor binding subsites +1 and +2 nearly converted the linkage specificity of Gtf-DSM to that of GtfO. Surprisingly, all of the individual or combinatorial mutations in four residues from domains IV and V of Gtf-DSM significantly altered the linkage specificity of Gtf-DSM. Additionally, all mutations in the 11 distinguishing residues of Gtf-DSM resulted in a dramatically reduced transferase/hydrolysis activity ratio, which was closer to that of GtfO. These mutation results suggested that the linkage specificity differences between Gtf-DSM and GtfO are determined by the distinct micro-physicochemical environments, formed by the concerted action of a series of residues not only from the acceptor binding subsites +1 and +2 but also from domains IV and V.

Structural basis for the linkage specificity of ubiquitin-binding domain and deubiquitinase.

Ubiquitination is a post-translational modification system essential for regulating a wide variety of biological processes in eukaryotes. Ubiquitin (Ub) itself undergoes post-translational modifications, including ubiquitination. All seven lysine residues and one N-terminal amino group of Ub can act as acceptors for further ubiquitination, producing eight types of Ub chains. Ub chains of different linkage types have different cellular functions and are referred to as the 'ubiquitin code'. Decoder molecules that contain linkage-specific Ub-binding domains (UBDs) recognize the Ub chains to regulate different cellular functions. On the other hand, deubiquitinases (DUBs) cleave Ub chains to reverse ubiquitin signals. This review discusses the molecular mechanisms of linkage-specific recognitions of Ub chains by UBDs and DUBs, which have been revealed by structural studies.

Novel Dextransucrase Gtf-DSM, Highly Similar in Sequence to Reuteransucrase GtfO, Displays Unique Product Specificity.

Lactic acid bacteria are known to produce extracellular polysaccharides such as α-glucan, levan, and inulin, which are promising for applications in food systems because of their prebiotic properties. In this work, a novel glucansucrase (GS) Gtf-DSM from Lactobacillus ingluviei DSM 14792 was recombinantly expressed and biochemically characterized as a dextransucrase capable of producing a dextran polysaccharide with four types of linkages, including 69% (α1 → 6), 24% (α1 → 3), 6% (α1 → 4), and 1% (α1 → 2). Intriguingly, the dextransucrase Gtf-DSM had a sequence identity of 99.3% with the reuteransucrase GtfO producing a reuteran with 21% (α1 → 6) and 79% (α1 → 4) linkages. Thus, the dextransucrase Gtf-DSM is a unique target for understanding the linkage specificity of GSs and the investigation of site-directed mutagenesis using Gtf-DSM and GtfO as templates is underway.

Stepwise multipolyubiquitination of p53 by the E6AP-E6 ubiquitin ligase complex.

The human papillomavirus (HPV) oncoprotein E6 specifically binds to E6AP (E6-associated protein), a HECT (homologous to the E6AP C terminus)-type ubiquitin ligase, and directs its ligase activity toward the tumor suppressor p53. To examine the biochemical reaction in vitro, we established an efficient reconstitution system for the polyubiquitination of p53 by the E6AP-E6 complex. We demonstrate that E6AP-E6 formed a stable ternary complex with p53, which underwent extensive polyubiquitination when the isolated ternary complex was incubated with E1, E2, and ubiquitin. Mass spectrometry and biochemical analysis of the reaction products identified lysine residues as p53 ubiquitination sites. A p53 mutant with arginine substitutions of its 18 lysine residues was not ubiquitinated. Analysis of additional p53 mutants retaining only one or two intact ubiquitination sites revealed that chain elongation at each of these sites was limited to 5-6-mers. We also determined the size distribution of ubiquitin chains released by en bloc cleavage from polyubiquitinated p53 to be 2-6-mers. Taken together, these results strongly suggest that p53 is multipolyubiquitinated with short chains by E6AP-E6. In addition, analysis of growing chains provided strong evidence for step-by-step chain elongation. Thus, we hypothesize that p53 is polyubiquitinated in a stepwise manner through the back-and-forth movement of the C-lobe, and the permissive distance for the movement of the C-lobe restricts the length of the chains in the E6AP-E6-p53 ternary complex. Finally, we show that multipolyubiquitination at different sites provides a signal for proteasomal degradation.

Profiling DUBs and Ubl-specific proteases with activity-based probes.

Protein (poly-)ubiquitination is a posttranslational modification that plays a key role in almost all cellular processes. It involves the installment of either single ubiquitin (Ub) moieties or one of eight different polyUb linkage types, each giving a distinct cellular outcome. Deubiquitinating enzymes (DUBs) reverse Ub signaling by disassembly of one or multiple poly-Ub chain types and their malfunction is often associated with human disease. The Ub system displays significant crosstalk with structurally homologous ubiquitin-like proteins (Ubls), including SUMO, Nedd8, and ISG15. This can be seen with the existence of heterogeneous chains made from Ub-Ubl mixtures as well as the proteolytic cross reactivity displayed by several DUBs toward other Ubl systems. In addition, numerous pathogens have been found to encode Ub(l)-ligases and deconjugating enzymes in order to facilitate infection and fight the host immune response. Studying the activity of DUBs and Ubl-specific proteases, both human as well as pathogen-derived, gives fundamental insights into their physiological roles. Activity-based probes (ABPs) have proven to be valuable tools to achieve this, as they report on enzyme activities by making a (often irreversible) covalent complex, rather than on their relative abundance. In this chapter, we explain the potential of ABPs to assess substrate preferences, structural features, and activity of Ub and Ubl deconjugating enzymes. We further demonstrate the practical use of ABPs to (1) characterize the activity of viral proteases toward Ub and Ubls and (2) to gain more insight in the structural determinants of substrate preference of DUBs.

Crystal structure and activity-based labeling reveal the mechanisms for linkage-specific substrate recognition by deubiquitinase USP9X.

USP9X is a conserved deubiquitinase (DUB) that regulates multiple cellular processes. Dysregulation of USP9X has been linked to cancers and X-linked intellectual disability. Here, we report the crystal structure of the USP9X catalytic domain at 2.5-Å resolution. The structure reveals a canonical USP-fold comprised of fingers, palm, and thumb subdomains, as well as an unusual ß-hairpin insertion. The catalytic triad of USP9X is aligned in an active configuration. USP9X is exclusively active against ubiquitin (Ub) but not Ub-like modifiers. Cleavage assays with di-, tri-, and tetraUb chains show that the USP9X catalytic domain has a clear preference for K11-, followed by K63-, K48-, and K6-linked polyUb chains. Using a set of activity-based diUb and triUb probes (ABPs), we demonstrate that the USP9X catalytic domain has an exo-cleavage preference for K48- and endo-cleavage preference for K11-linked polyUb chains. The structure model and biochemical data suggest that the USP9X catalytic domain harbors three Ub binding sites, and a zinc finger in the fingers subdomain and the ß-hairpin insertion both play important roles in polyUb chain processing and linkage specificity. Furthermore, unexpected labeling of a secondary, noncatalytic cysteine located on a blocking loop adjacent to the catalytic site by K11-diUb ABP implicates a previously unreported mechanism of polyUb chain recognition. The structural features of USP9X revealed in our study are critical for understanding its DUB activity. The new Ub-based ABPs form a set of valuable tools to understand polyUb chain processing by the cysteine protease class of DUBs.

Oligomerization of the HECT ubiquitin ligase NEDD4-2/NEDD4L is essential for polyubiquitin chain assembly.

The NEDD4-2 (neural precursor cell-expressed developmentally down-regulated 4-2) HECT ligase catalyzes polyubiquitin chain assembly by an ordered two-step mechanism requiring two functionally distinct E2∼ubiquitin-binding sites, analogous to the trimeric E6AP/UBE3A HECT ligase. This conserved catalytic mechanism suggests that NEDD4-2, and presumably all HECT ligases, requires oligomerization to catalyze polyubiquitin chain assembly. To explore this hypothesis, we examined the catalytic mechanism of NEDD4-2 through the use of biochemically defined kinetic assays examining rates of 125I-labeled polyubiquitin chain assembly and biophysical techniques. The results from gel filtration chromatography and dynamic light-scattering analyses demonstrate for the first time that active NEDD4-2 is a trimer. Homology modeling to E6AP revealed that the predicted intersubunit interface has an absolutely conserved Phe-823, substitution of which destabilized the trimer and resulted in a ≥104-fold decrease in kcat for polyubiquitin chain assembly. The small-molecule Phe-823 mimic, N-acetylphenylalanyl-amide, acted as a noncompetitive inhibitor (Ki = 8 ± 1.2 mm) of polyubiquitin chain elongation by destabilizing the active trimer, suggesting a mechanism for therapeutically targeting HECT ligases. Additional kinetic experiments indicated that monomeric NEDD4-2 catalyzes only HECT∼ubiquitin thioester formation and monoubiquitination, whereas polyubiquitin chain assembly requires NEDD4-2 oligomerization. These results provide evidence that the previously identified sites 1 and 2 of NEDD4-2 function in trans to support chain elongation, explicating the requirement for oligomerization. Finally, we identified a conserved catalytic ensemble comprising Glu-646 and Arg-604 that supports HECT-ubiquitin thioester exchange and isopeptide bond formation at the active-site Cys-922 of NEDD4-2.

The mechanism of neural precursor cell expressed developmentally down-regulated 4-2 (Nedd4-2)/NEDD4L-catalyzed polyubiquitin chain assembly.

The mechanism of Nedd4-2 has been quantitatively explored for the first time using biochemically defined kinetic assays examining rates of 125I-polyubiquitin chain assembly as a functional readout. We demonstrate that Nedd4-2 exhibits broad specificity for E2 paralogs of the Ubc4/5 clade to assemble Lys63-linked polyubiquitin chains. Full-length Nedd4-2 catalyzes free 125I-polyubiquitin chain assembly by hyperbolic Michaelis-Menten kinetics with respect to Ubc5B∼ubiquitin thioester concentration (Km = 44 ± 6 nm; kcat = 0.020 ± 0.007 s-1) and substrate inhibition above 0.5 µm (Ki = 2.5 ± 1.3 µm) that tends to zero velocity, requiring ordered binding at two functionally distinct E2∼ubiquitin-binding sites. The Ubc5BC85A product analog non-competitively inhibits Nedd4-2 (Ki = 2.0 ± 0.5 µm), consistent with the presence of the second E2-binding site. In contrast, the isosteric Ubc5BC85S-ubiquitin oxyester substrate analog exhibits competitive inhibition at the high-affinity Site 1 (Ki = 720 ± 340 nm) and non-essential activation at the lower-affinity Site 2 (Kact = 750 ± 260 nm). Additional studies utilizing Ubc5BF62A, defective in binding the canonical E2 site, demonstrate that the cryptic Site 1 is associated with thioester formation, whereas binding at the canonical site (Site 2) is associated with polyubiquitin chain elongation. Finally, previously described Ca2+-dependent C2 domain-mediated autoinhibition of Nedd4-2 is not observed under our reported experimental conditions. These studies collectively demonstrate that Nedd4-2 catalyzes polyubiquitin chain assembly by an ordered two-step mechanism requiring two dynamically linked E2∼ubiquitin-binding sites analogous to that recently reported for E6AP, the founding member of the Hect ligase family.

Functional Analysis of a Novel ß-(1,3)-Glucanase from Corallococcus sp. Strain EGB Containing a Fascin-Like Module.

A novel ß-(1,3)-glucanase gene designated lamC, cloned from Corallococcus sp. strain EGB, contains a fascin-like module and a glycoside hydrolase family 16 (GH16) catalytic module. LamC displays broad hydrolytic activity toward various polysaccharides. Analysis of the hydrolytic products revealed that LamC is an exo-acting enzyme on ß-(1,3)(1,3)- and ß-(1,6)-linked glucan substrates and an endo-acting enzyme on ß-(1,4)-linked glucan and xylan substrates. Site-directed mutagenesis of conserved catalytic Glu residues (E304A and E309A) demonstrated that these activities were derived from the same active site. Excision of the fascin-like module resulted in decreased activity toward ß-(1,3)(1,3)-linked glucans. The carbohydrate-binding assay showed that the fascin-like module was a novel ß-(1,3)-linked glucan-binding module. The functional characterization of the fascin-like module and catalytic module will help us better understand these enzymes and modules.IMPORTANCE In this report of a bacterial ß-(1,3)(1,3)-glucanase containing a fascin-like module, we reveal the ß-(1,3)(1,3)-glucan-binding function of the fascin-like module present in the N terminus of LamC. LamC displays exo-ß-(1,3)/(1,6)-glucanase and endo-ß-(1,4)-glucanase/xylanase activities with a single catalytic domain. Thus, LamC was identified as a novel member of the GH16 family.

Mechanisms of Deubiquitinase Specificity and Regulation.

Protein ubiquitination is one of the most powerful posttranslational modifications of proteins, as it regulates a plethora of cellular processes in distinct manners. Simple monoubiquitination events coexist with more complex forms of polyubiquitination, the latter featuring many different chain architectures. Ubiquitin can be subjected to further posttranslational modifications (e.g., phosphorylation and acetylation) and can also be part of mixed polymers with ubiquitin-like modifiers such as SUMO (small ubiquitin-related modifier) or NEDD8 (neural precursor cell expressed, developmentally downregulated 8). Together, cellular ubiquitination events form a sophisticated and versatile ubiquitin code. Deubiquitinases (DUBs) reverse ubiquitin signals with equally high sophistication. In this review, we conceptualize the many layers of specificity that DUBs encompass to control the ubiquitin code and discuss examples in which DUB specificity has been understood at the molecular level. We further discuss the many mechanisms of DUB regulation with a focus on those that modulate catalytic activity. Our review provides a framework to tackle lingering questions in DUB biology.

Chemical Synthesis of Activity-Based Diubiquitin Probes.

Activity-based diubiquitin probes are highly useful in probing the deubiquitinase (DUB) activity and ubiquitin chain linkage specificity. Here we describe a detailed protocol to synthesize a new class of diubiquitin DUB probes. In this method, two ubiquitin moieties are connected through a linker resembling the native linkage in size and containing a Michael acceptor for trapping the DUB active-site cysteine. Detailed procedures for generating the linker molecule are also described.

Characterization of the glucansucrase GTF180 W1065 mutant enzymes producing polysaccharides and oligosaccharides with altered linkage composition.

Exopolysaccharides produced by lactic acid bacteria are extensively used for food applications. Glucansucrase enzymes of lactic acid bacteria use sucrose to catalyze the synthesis of α-glucans with different linkage compositions, size and physico-chemical properties. Crystallographic studies of GTF180-ΔN show that at the acceptor binding sites +1 and +2, residue W1065 provides stacking interactions to the glucosyl moiety. However, the detailed functional roles of W1065 have not been elucidated. We performed random mutagenesis targeting residue W1065 of GTF180-ΔN, resulting in the generation of 10 mutant enzymes that were characterized regarding activity and product specificity. Characterization of mutant enzymes showed that residue W1065 is critical for the activity of GTF180-ΔN. Using sucrose, and sucrose (donor) plus maltose (acceptor) as substrates, the mutant enzymes synthesized polysaccharides and oligosaccharides with changed linkage composition. The stacking interaction of an aromatic residue at position 1065 is essential for polysaccharide synthesis.

The emerging role of deubiquitination in nucleotide excision repair.

Nucleotide excision repair (NER) protects genome stability by eliminating DNA helix distorting lesions, such as those induced by UV radiation. The addition and removal of ubiquitin, namely, ubiquitination and deubiquitination, have recently been demonstrated as general mechanisms to regulate protein functions. Accumulating evidence shows that several NER factors are subjected to extensive regulation by ubiquitination and deubiquitination. Thus, the balance between E3 ligases and deubiquitinating enzyme activities can dynamically alter the ubiquitin landscape at DNA damage sites, thereby regulating NER efficiency. Current knowledge about XPC ubiquitination by different ubiquitin E3 ligases highlights the importance of ubiquitin linkage types in regulating XPC binding and release from damaged DNA. Here, we discuss the emerging roles of deubiquitinating enzymes and their ubiquitin linkage specificities in NER.

The linkage specificity determination of Ube2g2-gp78 mediated polyubiquitination.

Polyubiquitin chain linkage specificity or topology is essential for its role in diverse cellular processes. Previous studies pay more attentions to the linkage specificity of the first ubiquitin moieties, whereas, little is known about the editing mechanism of linkage specificity in longer polyubiquitin chains. gp78 and its cognate E2-Ube2g2 catalyze lysine48 (K48)-linked polyubiquitin chains to promote the degradation of targeted proteins. Here, we show that the linkage specificity of the entire polyubiquitin chain is determined by the conjugation manner of the first ubiquitin molecule but not the following ones. Further study discovered that the gp78 CUE domain works as a proofreading machine during the growth of K48-linked polyubiquitin chains to ensure the linkage specificity. Together, our studies uncover a novel mechanism underlying the linkage specificity determination of longer polyubiquitin chains.