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OTUB1 binds Ubiquitin. 83 / 99
| 12 71
sparser
"The interaction of donor Ub with the N-terminal helix of OTUB1 is strikingly reminiscent of the interaction between Ub interaction motifs (UIMs) and Ub ( xref )."
22325355
sparser
"These results suggest that the N-terminus of OTUB1 interferes with UEV binding and thus with K63Ub synthesis, and that the ability of the N-terminus to interfere with UEV depends upon a conformational change that is triggered by binding of free ubiquitin to OTUB1."
22367539
sparser
"The donor ubiquitin binds to the proximal site of OTUB1 ( xref ) in an orientation that places K48 of the ubiquitin near the OTUB1 active site ( xref )."
22367539
sparser
"Ubal binds to the distal ubiquitin binding site of OTUB1 ( xref ) in a manner similar to yeast xref and viral xref – xref OTU enzymes, and accounts for the effects of mutations in the OTUB1 distal site ( xref )."
22367539
reach
"OTUB1 binds preferentially to the Ub charged E2 in a manner that requires an N-terminal extension to its core OTU domain."
22325355
reach
"Surprisingly, we found that in addition to binding the E2 ~ Ub, OTUB1 also binds free Ub."
22325355
reach
"Perhaps the most surprising feature of the structure was the presence of a non covalently linked Ub molecule bound to OTUB1 (XREF_FIG, XREF_FIG, XREF_SUPPLEMENTARY)."
22325355
reach
"Further demonstrating an interdependency of binding, we found that free Ub bound to OTUB1 only in the presence of UbcH5b ~ Ub (XREF_FIG)."
22325355
reach
"If one considers that OTUB1 binds primarily to the Ub charged form of E2s, these OTUB1 levels are sufficient to regulate UBC13 action and the DSB response."
22325355
reach
"This affords van der Waals and salt bridge (between Glu51 in donor Ub and Arg72 and Arg74 in free Ub) interactions between Ub molecules (XREF_FIG), which we suspected could confer interdependency of Ub ~ UbcH5b and Ub binding to OTUB1."
22325355
reach
"To further probe the importance of free Ub binding to OTUB1, we designed an E195R mutation, which we predicted to potently impair binding to free Ub."
22325355
sparser
"OTUB1 binding to the charged E2~Ub is allosterically regulated by an additional free ubiquitin monomer that binds to the OTUB1 distal ubiquitin-binding site, triggering conformational changes that favor binding of the UBC13~Ub donor ubiquitin in the OTUB1 proximal site xref ."
23955022
sparser
"The requirement for the binding of a free ubiquitin monomer to OTUB1 raised the interesting possibility that changes in cellular ubiquitin concentrations might regulate the ability of OTUB1 to inhibit E2 enzymes."
23955022
sparser
"Comparisons of Ub-bound Otu1 to the OTU domain of human DUB Otubain 2 alone revealed a disordered loop, which is positioned to sterically clash with a bound Ub."
33081638
sparser
"The repressive complex formed by OTUB1 binding to charged E2~Ub and free ubiquitin presumably interferes with OTUB1 DUB activity by occupying both proximal and distal ubiquitin-binding sites of OTUB1, thus precluding binding of a Lys48 polyubiquitin substrate."
23955022
sparser
"In Ub-bound Otu1, this region forms an ordered β-strand, suggesting that Ub binding to Otu1 may induce a conformation change that stabilizes the disordered region in a β-strand, removing this steric hindrance [ xref ]."
33081638
reach
"In recent complex structures of OTUB1 with Ub bound in the S1 ' site of the DUB and of OTULIN bound to Met1 linked diUb, additional N-terminal helices form extensive S1 ' sites (XREF_FIG J, 5K, XREF_FIG I, and S5J)."
23827681
sparser
"Perhaps the most surprising feature of the structure was the presence of a non-covalently linked Ub molecule bound to OTUB1 ( xref , xref , xref )."
22325355
sparser
"The OTU deubiquitinase ubiquitin aldehyde-binding 1 (OTUB1) is commonly found in amyloid plaques associated with AD [ xref , xref ]."
39562545
sparser
"Examining the extant structures of OTUB1 bound to ubiquitin aldehyde and a chemical UBE2D2 ∼ ubiquitin conjugate reveals that a substrate attached to the proximal subunit of a K48 chain could sterically block the chain from interacting with OTUB1. xref "
30137960
sparser
"Based on this interpretation, we hypothesized that UBCH5B would not stimulate OTUB1 cleavage of ubiquitin-AMC, a substrate that binds to the distal ubiquitin-binding site of OTUB1 but contains an amide-linked fluorophore (amino methyl coumarin) in place of a proximal ubiquitin."
23955022
sparser
"These residues, which are N-terminal to the OTU catalytic domain, are disordered in the apoenzyme xref , xref but form an alpha helix when OTUB1 is bound to both distal ubiquitin and charged UBC13~Ub xref or UBCH5B~Ub xref ."
23955022
sparser
"In the absence of a structure of OTUB1 bound to Lys48 diubiquitin and E2, we took advantage of the observation that the two ubiquitins bound to the proximal and distal sites of OTUB1 mimic the binding of Lys48 diubiquitin xref , xref to examine the structural effect of E2 binding on the N-terminal ubiquitin binding helix of OTUB1."
23955022
sparser
"The complete repression of OTUB1 activity at >10 µM free ubiquitin is presumably due to formation of the inhibited OTUB1-Ub-UBCH5B~Ub complex ( xref ), in which free ubiquitin binds to the OTUB1 distal ubiquitin-binding site while the ubiquitin conjugated to UBCH5B binds to the OTUB1 proximal site, thus precluding binding of the Lys48 diubiquitin substrate."
23955022
sparser
"The structural and energetic coupling between these different binding interactions thus drives formation of the different OTUB1-E2-ubiquitin complexes."
23955022
sparser
"A charged E2~Ub conjugate, however, will bind to OTUB1 with the conjugated donor ubiquitin bound in the OTUB1 proximal site, while free ubiquitin binds to the OTUB1 distal site and allosterically stimulates binding of OTUB1 to the E2~Ub conjugate."
23955022
sparser
"Accumulation of Lys48 polyubiquitin could further tip the balance in favor of the active DUB complex by competing with charged E2~Ub thioester for binding to the ubiquitin-binding sites of OTUB1 ( xref )."
23955022
sparser
"The affinity of OTUB1 for the charged E2-Ub is increased when a free ubiquitin monomer binds to the distal site of OTUB1 [ xref , xref , xref ]."
33498168
sparser
"Also, free ubiquitin binding to the distal site of OTUB1 has been shown to compete with the binding of K48-linked chains and inhibits the deubiquitinating activity of OTUB1 [ xref ]."
33498168
sparser
"The N-terminal residues of the OTU domain in OTUB1 are required for binding to UBC13 ~ Ub and this interaction is facilitated by the binding of a free Ub to a second site in OTUB1, resulting in allosteric change in the OTU domain and the formation of a ubiquitin-binding helix in the N-terminus which increase its affinity for UBC13-Ub."
28031783
sparser
"These include interaction of the E2 with an N-terminal alpha helix and the C-terminus of OTUB1, interaction of the donor ubiquitin (ubiquitin conjugated to the E2) with the N-terminal alpha helix and OTU core of OTUB1 and, unexpectedly, interaction of a free ubiquitin with the C-terminus of OTUB1."
30400005
sparser
"Expanding on this study, Weiner and colleagues found that free ubiquitin binding to OTUB1’s C-terminus allosterically triggers the formation of the E2/ubiquitin binding alpha helix in OTUB1’s N-terminus and that this is essential for E2 inhibition."
30400005
sparser
"In addition, in vitro work demonstrated that in the absence of free ubiquitin, OTUB1 does not bind to ubiquitin-charged E2s and that free ubiquitin enhances E2 binding in a concentration dependent manner ( xref )."
30400005
sparser
"Free ubiquitin can bind to a second site in OTUB1 and increase the affinity with UBC13, preventing ubiquitin transfer and suppressing the accumulation of polyubiquitin [ xref , xref , xref ]."
37632103
sparser
"B1.1 was identified as an ubiquitin mutant that bound to the distal ubiquitin binding site of OTUB1 and was demonstrated to inhibit OTUB1 catalytic activity and interfere with OTUB1-E2 complex formation."
30400005
sparser
"Because in a previously published co-crystal structure recognition of noncyclic Ub 2 by OTUB1 was shown to be asymmetric, i.e., the proximal ubiquitin subunit associated with OTUB1 via a larger surfac[MISSING/INVALID CREDENTIALS: limited to 200 char for Elsevier]"
34049206
sparser
"Here we elucidate the structural mechanism by which OTUB1 binds E2s to inhibit ubiquitin transfer."
22325355
sparser
"Surprisingly, free ubiquitin associates with the canonical distal ubiquitin-binding site on OTUB1 to promote formation of the inhibited E2 complex."
22325355
sparser
"OTUB1 binds preferentially to the Ub-charged E2 in a manner that requires an N-terminal extension to its core OTU domain ( xref ; xref )."
22325355
sparser
"B1.1 was identified as a ubiquitin mutant that binds to the distal ubiquitin binding site of OTUB1, effectively inhibiting its catalytic activity and interfering with OTUB1-E2 complex formation [ xref ]."
39562545
sparser
"Surprisingly, we found that in addition to binding the E2~Ub, OTUB1 also binds free Ub."
22325355
sparser
"Together, these studies provide new insights into ubiquitin binding and hydrolysis by yeast Otu1 and other OTU domain-containing proteins."
18270205
sparser
"This affords van der Waals and salt bridge (between Glu51 in donor Ub and Arg72 and Arg74 in free Ub) interactions between Ub molecules ( xref ), which we suspected could confer interdependency of Ub~UbcH5b and Ub binding to OTUB1."
22325355
sparser
"Further demonstrating an interdependency of binding, we found that free Ub bound to OTUB1 only in the presence of UbcH5b~Ub ( xref )."
22325355
sparser
"Additionally, analysis of the structure of the OTUB1-Ub (PDB:) complex xref revealed that Thr7 is located in the interface between OTUB1 and distal ubiquitin, while the residues Thr12 and Thr66 are solvent-exposed ()."
40443892
sparser
"OTUB1 binds free ubiquitin, which induces conformational changes in the catalytic domain to allow simultaneous binding of the charged ubiquitin on the E2."
25906909
sparser
"The CCHFV-OTU X-ray structure in complex with Ub revealed that the N-terminal β-strands interact with a hydrophobic patch in Ub, resulting in a −75° rotation of Ub with relationship to the OTU protease, as compared to OTUB1-Ub complex xref ."
| PMC
sparser
"Interestingly, ubiquitin binding to ZnF4/7 domains but not ubiquitin-hydrolyzing activity by the OTU domain facilitates A20 auto-ubiquitination with degradative K48-linked ubiquitin chains for directing A20 to the proteasomal pathway (Fig.  xref H)."
35099607
sparser
"Thus, the current structure of OTUB1 bound to ubiquitin does not provide an explanation for the inhibitory effect of Thr12 and Thr66 phosphorylation on OTUB1-catalyzed K48-linked diubiquitin cleavage."
40443892
sparser
"Although this S1 site is on a similar surface of the OTU domain, the distinctive recognition elements (to be discussed in a broader context below) position the bound Ub moiety in a drastically different orientation that is 107° or 167° rotated from the vOTU‐Ub or OTUB1:Ub structures, respectively (Fig  xref D; Akutsu et al , xref ; Juang et al , xref )."
32567101
reach
"In addition, OTUB1-C23-SSG or C204-SSG could realign the OTUB1-bound UBCH5 and free Ub, into a configuration that increases the potency of OTUB1-mediated inhibition of ubiquitination."
37703360
sparser
"OTUB1 can also simultaneously bind free Ub and certain E2∼Ub conjugates (e.g., Ube2D(1/2/3)∼Ub, Ube2E1∼Ub, or Ube2N∼Ub)."
27002219
sparser
"The Ub conjugated to UbcH5b predominately interacts with OTUB1; one of these interactions is mediated by the N-terminus of OTUB1 discussed above, which forms an extended helix ( xref )."
23845989
sparser
"The binding of a free ubiquitin at the distal site allosterically triggers a conformational change in the OUT domain and increases the OTUB1 binding affinity to the donor ubiquitin, thus promoting the binding of OTUB1 and donor ubiquitin and disrupting the E2-ubiquitin interaction."
23234564
sparser
"We also examined the effect of S65 phosphorylation on Ub binding to K48-specific deubiquitinase OTUB1, which interacts with the hydrophobic patch surface on Ub or Ub 2 xref ."
29422536
sparser
"Structural studies of these complexes [ xref , xref ] reveal that the donor ubiquitin binds an otherwise disordered N-terminal segment of OTUB1 in a manner that is facilitated by binding of a second, free ubiquitin."
23896398
sparser
"The structure of yeast OTU1 bound to a ubiquitin based inhibitor has been solved and comparison with the free structure of human Otubain-2 suggests that the OTU domain also may exhibit active site rearrangements upon binding to ubiquitin ( xref )."
19489724
reach
"Unexpectedly a second, free Ub was bound to OTUB1 (S1 site) and its C-terminal tail was juxtaposed near K48 of UbcH5 conjugated Ub within the catalytic cleft [XREF_BIBR]."
23845989
reach
"Another notable finding from this study is that free Ub binding to OTUB1 (at S1) allosterically regulates the enzyme by increasing its affinity for Ubc13 ~ Ub (at S1 ') [XREF_BIBR]."
23845989
sparser
"Thus OTUB1 simultaneously binds to E2-charged Ub and a free Ub, and the arrangement of these two ubiquitins mimics K48 di-Ub."
23845989
sparser
"To accommodate structural differences between Ub and ISG15, the viral protease binds the β-grasp folds of Ub and C-terminal Ub-like domain of ISG15 in an orientation that is rotated nearly 75° with respect to that observed for Ub bound to a representative eukaryotic OTU domain from yeast."
21245344
sparser
"Interaction of the OTUB1 N-terminus with uncharged E2s (such as UBCH5B) increases OTUB1 activity towards K48-ubiquitin chains by stabilizing the structure of an OTUB1 Ub-binding site [ xref ]."
31299612
sparser
"Another notable finding from this study is that free Ub binding to OTUB1 (at S1) allosterically regulates the enzyme by increasing its affinity for Ubc13~Ub (at S1’) [ xref ]."
23845989
sparser
"The interface of our query chain was aligned with the interface of ubiquitin (chain D) in this complex of human ubiquitin and the ubiquitin thioesterase OTU1."
38470439
sparser
"Surprisingly, OTUB1 simultaneously binds a ubiquitin-charged E2 enzyme and a free ubiquitin molecule, thereby adopting a conformation that mimics the cleavage product of a Lys48-linked ubiquitin chain[MISSING/INVALID CREDENTIALS: limited to 200 char for Elsevier]"
22464734
sparser
"B1.1 also obviously restrains OTUB1 activity by binding to the terminal ubiquitin binding site of OTUB1, further cutting off the K48 linked ubiquitin and disturbing the formation of OTUB1-E2 complex [[MISSING/INVALID CREDENTIALS: limited to 200 char for Elsevier]"
34875341
sparser
"This process involves the binding of the distal ubiquitin to the catalytic site of OTUB1, triggering a conformational change that enables the binding of the proximal ubiquitin to the N-terminal region of OTUB1, along with the proper positioning of the K48 scissile bond for cleavage ( xref )."
38455765
sparser
"NMR chemical shift perturbation studies and activity assays with mutant diUbs indicate that the proximal and distal Ubs bind to separate sites on hOtu1."
19211026
sparser
"This process is allosterically regulated by free ubiquitin, which binds to the distal site of OTUB1 and enhances its affinity for E2 enzymes that are covalently linked to ubiquitin."
38455765
reach
"Mechanistically, OTUB1 interacts with and inhibits several E2 Ub-conjugating enzymes, including UBC13 and UBCH5."
35764170
sparser
"For activation of the inhibitory function, free ubiquitin must bind to the distal ubiquitin-binding site of OTUB1 [ xref ]."
26983989
sparser
"Because the N-terminal residues are usually disordered, OTUB1 cannot interact with ubiquitin that is charged on UBC13."
26983989
sparser
"The binding of free ubiquitin to the distal ubiquitin binding site of OTUB1 triggers conformational changes in the OTU domain and the formation of a ubiquitin-binding helix in the N terminus of OTUB1, promoting tight interaction between OTUB1 and ubiquitin-charged UBC13 [ xref ]."
26983989
sparser
"This allosteric regulation of OTUB1 suggests that a locally highly elevated concentration of free ubiquitin, which can be produced by the deubiquitination of ubiquitinated proteins at DSB sites, promotes interaction between OTUB1 and ubiquitin-charged E2 conjugating enzymes."
26983989
sparser
"B1.1 binds to the distal Ub-binding site of OTUB1 ( xref ) and enables a rationalization of its altered binding and allosteric properties."
23287719
sparser
"The previous crystallographic analyses show that the residues amino-terminal to the OUT domain of OTUB1 (85–98 aa) are required for binding to UBC13∼Ub and inhibition of K63Ub synthesis ( xref ), free ubiquitin binding to OTUB1's C-terminus allosterically triggers the formation of the E2/ubiquitin–binding alpha helix in OTUB1's N-terminus and that is essential for E2 inhibition ( xref ), and deletion of the first 45 residues of OTUB1 abolishes the ability of OTUB1 to noncanonically inhibit E2s ( xref )."
36822329
sparser
"Crystal structures of an OTUB1-UBC13 complex and of OTUB1 bound to ubiquitin aldehyde and a chemical UBC13~Ub conjugate show that binding of free ubiquitin to OTUB1 triggers conformational changes in the OTU domain and formation of a ubiquitin-binding helix in the N-terminus, thus promoting binding of the conjugated donor ubiquitin in UBC13~Ub to OTUB1."
22367539
sparser
"It was previously shown that binding of the covalent inhibitor, ubiquitin aldehyde (Ubal), to the distal ubiquitin-binding site of OTUB1 stimulates binding of ubiquitin vinyl sulfone to N-terminus xref ."
22367539
sparser
"Since the OTUB1 N-terminus was implicated in binding to the donor ubiquitin in the UBC13~Ub conjugate xref , we asked whether Ubal binding to OTUB1 could enhance inhibition of UBC13 by stimulating binding of the OTUB1 N-terminus to the donor ubiquitin."
22367539
sparser
"This prompted us to ask whether free ubiquitin binding to the OTUB1 distal site could similarly stimulate binding of OTUB1 to UBC13~Ub conjugates."
22367539
sparser
"To verify that ubiquitin binding to the distal site of OTUB1 is important for inhibition of UBC13, we assayed the effect of distal site mutations, which were chosen based on structures of a covalent yOTU1-ubiquitin complex xref and of human OTUB1 xref ."
22367539
sparser
"Taken together, our results indicate that the ability of OTUB1 to bind preferentially to the UBC13~Ub conjugate and inhibit ubiquitin transfer is allosterically regulated by free ubiquitin binding to the distal site of OTUB1, which triggers capture of the conjugated ubiquitin in the OTUB1 proximal site."
22367539
sparser
"Furthermore, OTUB1 (OTU deubiquitinase ubiquitin aldehyde-binding 1) was found in Aβ plaques of AD patients [ xref ], and interestingly, USP14 was able to inhibit tau function [ xref ]."
| PMC