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USP12 binds WDR48. 134 / 137
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"(G) Superposition of the free and UAF1-bound USP12 structures focusing on the Palm domain, the catalytic cleft, BL2, and BL3."
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"In the UAF1-USP12 complex structure, this aromatic residue is dislodged from the pocket but tucked underneath the Ring Finger between the side chains of Arg245 and Lys247 ( Figure 3D )."
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"We then show that in solution USP12 can bind to a second molecule UAF1, but with lower affinity."
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"Reciprocal IP-MS/MS suggests the existence of multiple distinct Dub complexes in which USP12 (or USP46) interacts with WDR48, WDR20 (or DMWD), and PHLPP (or PHLPPL) (XREF_FIG, XREF_SUPPLEMENTARY)."
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"Overall Architecture of the UAF1-USP12 Complex (A and B) Ribbon diagram of the UAF1-USP12 complex in two orthogonal views."
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"Overall, our two UAF1-USP12 structures not only reveal a series of structural changes of the enzyme that are connected to the binding site of UAF1 but also highlight the dynamic nature of three key loops, namely, BL2, BL3, and the CC Loop, surrounding the catalytic site of the activated enzyme."
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"Interestingly, the addition of WDR20 to the USP12 and UAF1 complex further increased USP12 's DUB activity."
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"The crystal structures of USP1-UAF1 reveal plasticity in USP1 and key differences to USP12-UAF1 and USP46-UAF1, two related proteases."
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"F219 and its two interacting residues in the UAF1-bound USP12 structure are highlighted in sticks."
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"To confirm that these structural differences of USP12 are associated with UAF1 binding but not crystal packing or resolution limit, we crystallized the UAF1 and USP12 complex in another space group (C[MISSING/INVALID CREDENTIALS: limited to 200 char for Elsevier]"
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"Although BL1 is destabilized in the crystal structures of the UAF1 and USP12 complex, it is expected to adopt a productive conformation upon substrate binding and play an important role in adjusting the topology of the Ub tail by fine tuning its preceding region."
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"We have previously demonstrated that Usp12 and Uaf -1 complex increases AR transcriptional activity."
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"Overall Architecture of the UAF1-USP12 Complex (A and B) Ribbon diagram of the UAF1-USP12 complex in two orthogonal views."
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"Binding of WDR20 to the UAF1 and USP12 complex induces multiple structural changes in the enzyme, converting it into a compact form similar to Ub bound USP46."
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"As both Uaf-1 and WDR20 interact with Usp12 we hypothesised that Uaf-1 and WDR20 would also be found in a complex with AR."
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"In addition to binding viral E1, UAF1 interacts with three deubiquitinating enzymes, USP1, USP12, and USP46, leading to formation of a ternary complex on the viral origin, consisting of E1, UAF1, and any one of the three DUBs [XREF_BIBR]."
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"In the UAF1 and USP12 complex structure, this aromatic residue is dislodged from the pocket but tucked underneath the Ring finger between the side chains of Arg245 and Lys247 (XREF_FIG)."
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No evidence text available
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"Of notice, even though BL1 of USP12 is completely collapsed in our UAF1 and USP12 complex structures, Ub binding is expected to stabilize it via substrate induced fit, as seen in other USPs and the Ub conjugated USP46 structures."
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"The structurally regularized Pinky Finger in UAF1-bound USP12 is indicated in (D)."
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"Furthermore, forced USP12 expression significantly potentiated the protein stability of PPM1B (Supplementary Fig. 3d), and the knockdown of WDR48, a binding partner of USP12 that was reported to contribute to USP12 deubiquitinase activity , suppressed PPM1B expression (Supplementary Fig. 3e)."
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"Even though BL1 of USP12 is collapsed in our UAF1-USP12 complex structures, Ub binding is expected to stabilize it via substrate-induced fit, as seen in other USPs and the Ub-conjugated USP46 structures."
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"Green dotted lines represent disordered ends of polypeptide breaks in the UAF1-bound USP12 structure (determined in the C2 space group)."
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"The UAF1-USP12 structure was determined with a combination of molecular replacement (MR) and single-wavelength anomalous diffraction methods."
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"In the two UAF1 and USP12 complex structures, UAF1 binding appears to induce BL1 destabilization via a chain of conformational changes, whereas the WDR20-USP12 interaction seems to overwrite the allosteric effect of UAF1 by re-stabilizing the loop."
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No evidence text available
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"Different from the UAF1-USP46 ~ Ub structure, the previously predicted C-terminal SUMO like domain of UAF1 is clearly resolved in the UAF1 and USP12 complex, in addition to the N-terminal WD40-repeat domain and a central ancillary domain (XREF_FIG, XREF_FIG, XREF_FIG, XREF_SUPPLEMENTARY)."
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"For example, WDR48 interacts with USP1, USP12, and USP46 to activate their DUB activity (Cohn et al., 2009; Yin et al., 2015)."
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"The partial overlap of the binding interfaces and the high affinity of WDR20 suggests that WDR20 binding to USP12 and UAF1 can either prevent binding of the second UAF1 molecule or it can actively compete out UAF1 if it is bound to the USP12 and UAF1 complex."
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No evidence text available
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"In the UAF1 and USP12 complex structure, this aromatic residue is dislodged from the pocket but tucked underneath the Ring Finger between the side chains of Arg245 and Lys247."
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"Usp12, Uaf-1 and WDR20 form a complex acting within a positive feedback loop in PC cells."
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No evidence text available
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"This latter possibility is surprising given that USP12 and 46 also bind WDR48, but lack this insertion."
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"We then show that in solution USP12 can bind to a second molecule UAF1, but with lower affinity."
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"For example, WDR48 interacts with USP1, USP12 and USP46 to activate their DUB activity."
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"Superposition analysis predicts that this third Palm domain loop is in collision with the Ub tail in the C2 space group UAF1-USP12 structure (Figure S3G) ."
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"In contrast, the PK Loop in the UAF1-USP12 complex structure appears to adopt a flexible structure, as evidenced by the absence of its electron density."
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"Despite a potential salt bridge and a small number of van der Waal interactions between the two b propellers, UAF1 binding to USP12 has little effect on the association of WDR20 with the enzyme (Figures S4A and S4B )."
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"Despite a potential salt bridge and a small number of van der Waal interactions between the two b propellers, UAF1 binding to USP12 has little effect on the association of WDR20 with the enzyme (Figures S4A and S4B )."
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No evidence text available
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"Despite a potential salt bridge and a small number of van der Waal interactions between the two beta propellers, UAF1 binding to USP12 has little effect on the association of WDR20 with the enzyme."
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"In C. elegans, USP46 and USP12 both bind to USP1 associated factor 1 (UAF1 and WDR48) and the binding dramatically enhances the activity of USP12 and USP46."
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"In the free USP12 structure and in the F222 space group structure of the UAF1-USP12 complex, this loop is excluded from the catalytic cleft by BL1 and BL2."
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"Despite a potential salt bridge and a small number of van der Waal interactions between the two β-propellers, UAF1 binding to USP12 has little effect on the association of WDR20 with the enzyme ( xref )."
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"(H) Interactions of wild-type (WT) and mutant USP12 with UAF1 assessed by immunoprecipitation (IP) and western blot using an in vitro transcription and translation system.the UAF1-USP12 complex, in addition to the N-terminal WD40repeat domain and a central ancillary domain(Figures 2A, 2B, 2D, and S2A-S2E)Structural Differences between Free and UAF1-Bound USP12(A) Side view of the interface between UAF1 (orange) and the tip of USP12 Pinky Finger (green)."
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"Our structures reveal that UAF1 and WDR20 interact with USP12 at two distinct sites far from its catalytic center."
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"The second binding of UAF1 to USP12 does not seem to play a role in the activation of USP12 on a minimal substrate."
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No evidence text available
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"In stark contrast, the PK Loop in the UAF1 and USP12 complex structure appears to adopt a flexible structure as evidenced by the absence of its electron density."
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"Reminiscent of the Ub-bound UAF1-USP46 complex, the UAF1-USP12 structure features an L-shaped architecture, in which the elongated UAF1 protein packs perpendicularly against the DUB (Figures 2A and 2B) ."
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"Binding of WDR20 to the UAF1-USP12 complex induces multiple structural changes in the enzyme, converting it into a compact form similar to Ub-bound USP46."
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"Such plasticity is detected crystallographically for BL2 and the CC Loop in our two UAF1-USP12 structures."
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"UAF1 binds USP12 in two distinct steps with different affinities."
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"By contrast, a comparison between free and UAF1 bound USP12 structures without suicidal Ub unveils a concatenation of conformational differences spreading across the protein."
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"Since the conformational change as well as the presence of two UAF1 interfaces seems to be conserved between USP46 and USP12, we wondered what stoichiometry the USP12 and UAF1 complex has in solution."
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"While the Pinky Finger of USP12 in the UAF1-USP12-WDR20 complex adopts the same b strand conformation as seen in the UAF1-USP12 complex, F219 on the PK Helix becomes embedded in the pocket formed between the Fingers and the Palm domains, instead of being tucked underneath the Fingers domain ( Figures 3D, 5B , and 5C)."
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"XREF_BIBR Notably, a recent study that screened a panel with 22 deubiquitinating enzymes in vitro showed that galeterone inhibits the enzymatic activity of USP12 and also binds directly to the USP12 and USP12 and UAF1 complexes, which dephosphorylate AKT."
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"WDR20 preserves and regulates the activity of the USP12-UAF1 deubiquitinating enzyme complex (87)."
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No evidence text available
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"Structural Comparison of Free and UAF1-bound USP12."
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"WDR48, a WD40 repeat containing protein, interacts with USP12 and USP46 and is required for the histone deubiquitination activity."
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"The k cat and K M values are presented as means ± SD (n = 3), and the K D values were obtained through steady-state analysis (see Supplemental Experimental Procedures for technical discussion). (B) Time course of USP12 modification by the suicidal Ub-VME substrate in the absence or presence of UAF1 and WDR20. (C) Time course of LRGG-AMC (top) in contrast to Ub-AMC (bottom) hydrolysis by USP12 (green), UAF1-USP12 (orange), and USP12-WDR20 (red)."
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"The USP12 BL1 residue F262 is shown in sticks to highlight its blocking position.Biochemical Analysis of USP12 Activation by UAF1 and WDR20 (A) Enzyme kinetics analyses of free and activator-bound USP12 with Ub-AMC substrate and binding analysis of a Ub(AKA) peptide to all forms of USP12, as measured by an Octet Bio-Layer Interferometry system."
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"In the free USP12 structure and in the F222 space group structure of the UAF1-USP12 complex, this loop is excluded from the catalytic cleft by BL1 and BL2."
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"In the two UAF1-USP12 complex structures, UAF1 binding appears to induce BL1 destabilization via a chain of conformational changes, whereas the WDR20-USP12 interaction seems to overwrite the allosteric effect of UAF1 by (D and E) Rates of Ub-AMC hydrolysis by wild-type (WT) and mutant USP12 in the presence of UAF1 (D) or WDR20 (E)."
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"Recently, our work led to the identification of the deubiquitinating complex USP12 and UAF1, which is recruited by Itch to non activated Notch and allows Notch deubiquitination, probably before its entry into the intraluminal vesicles of the MVBs [XREF_BIBR]."
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"All mutants impaired UAF1 and USP12 complex formation (XREF_FIG and XREF_SUPPLEMENTARY)."
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"Although BL1 is destabilized in the crystal structures of the UAF1-USP12 complex, it is expected to adopt a productive conformation upon substrate binding and play an important role in adjusting the topology of the Ub tail by fine-tuning its preceding region."
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"Different from the UAF1-USP46 ~ Ub structure, the previously predicted C-terminal SUMO like domain of UAF1 is clearly resolved in the UAF1 and USP12 complex, in addition to the N-terminal WD40-repeat [MISSING/INVALID CREDENTIALS: limited to 200 char for Elsevier]"
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"The dashed lines in (F) indicate the disordered PK Loop and BL1 in the UAF1-bound USP12 structure."
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"Overall, the UAF1, USP12, and WDR20 complex is distinguished from the two free USP12 structures and the two UAF1 and USP12 complex structures by the apparent stabilization of most flexible structural [MISSING/INVALID CREDENTIALS: limited to 200 char for Elsevier]"
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No evidence text available
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"All mutants impaired UAF1 and USP12 complex formation."
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"This model is inferred from a combination of structures of USP46 bound to WDR48 and ubiquitin (Yin et al., 2015) and USP12 bound to WDR48 and WDR20 (Li et al., 2016)."
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"All mutants impaired UAF1-USP12 complex formation ( Figures 2H and S2G )."
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No evidence text available
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"Our structures reveal that UAF1 and WDR20 interact with USP12 at two distinct sites far away from its catalytic center."
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"To confirm that these structural differences of USP12 are associated with UAF1 binding but not crystal packing or resolution limit, we crystallized the UAF1 and USP12 complex in another space group (C2) and determined its structure at 2.3 A resolution."
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"Here, we report the crystal structure of the USP12-Ub and UAF1 complex at a resolution of 2.8 A and of UAF1 at 2.3 A."
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"The second binding of UAF1 to USP12 does not seem to play a role in the activation of USP12 on a minimal substrate."
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"In contrast, the PK Loop in the UAF1 and USP12 complex structure appears to adopt a flexible structure, as evidenced by the absence of its electron density."
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"Sticks show the disorganized catalytic center in the UAF1-bound USP12 structure.Crystal Structure of a UAF1-USP12-WDR20 Complex (A) Ribbon diagram of the UAF1DSLD-USP12-WDR20 complex with WDR20 shown in a slate color and the catalytic triad in sticks."
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"UAF1 also binds and activates two other DUBs, USP12 and USP46 (19), and studies that reveal how UAF1 binds and activates USP12 and USP46 suggest that UAF1 will bind to USP1 in an analogous manner (20, 21)."
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"In this regard, it has been reported that, besides USP1, UAF1 also binds and regulates the activity of two other DUBs, namely USP12 and USP46 XREF_BIBR."
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"The accession numbers for the coordinates and structure factors reported in this paper are PDB: 5K16, 5K1B, 5K1A, 5K19, and 5K1C for free USP12, UAF1-USP12 (F222 space group), UAF1-USP12 (C2 space group), WDR20, and UAF1-USP12-WDR20, respectively."
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"3.3 UAF1 binds USP12 in two distinct steps with different affinities."
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"We validated the UAF1-USP12 interface by mutating two neighboring residues (Q240 and E241) at the center of the USP12 fingertip region and two pairs of aromatic residues at the top surface of the UAF1 WD40-repeat domain ( Figures 2F and S2F )."
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"In the UAF1-USP12 complex structure, this aromatic residue is dislodged from the pocket but tucked underneath the Ring Finger between the side chains of Arg245 and Lys247 ( Figure 3D )."
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"Even though BL1 of USP12 is collapsed in our UAF1 and USP12 complex structures, Ub binding is expected to stabilize it via substrate induced fit, as seen in other USPs and the Ub conjugated USP46 stru[MISSING/INVALID CREDENTIALS: limited to 200 char for Elsevier]"
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"A defining feature of WDR20‐dependent activation is that it is necessary for the ability of USP12 to cleave a peptide substrate, Leu‐Arg‐Gly‐Gly (LRGG)‐AMC, since USP12‐UAF1 alone cannot cleave such a substrate (Li et al , xref )."
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"To confirm that these structural differences of USP12 are associated with UAF1 binding but not crystal packing or resolution limit, we crystallized the UAF1-USP12 complex in another space group (C2) and determined its structure at a 2."
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"By contrast, a comparison between the free and UAF1 bound USP12 structures without a suicidal Ub unveils a concatenation of conformational differences spreading across the protein."
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"Although the active site of USP12 in the UAF1-USP12 complex crystallized in F222 space group adopts the same topology as found in the free enzyme ( Figure S3F ), in the C2 space group structure, the USP12 Thumb domain features a series of major structural rearrangements at the catalytic cleft."
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"Structural Comparison of Free and UAF1 bound USP12."
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"The human UAF1 or UAF1 and USP12 complex was expressed and purified from Hi5 insect cells."
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"The dashed line indicates the disordered PK Loop of UAF1-bound USP12."
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"The dashed line indicates the disordered PK Loop of UAF1-bound USP12."
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"All mutants impaired UAF1-USP12 complex formation ( Figures 2H and S2G )."
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"UAF1 also interacts with DUBs USP12 and USP46 (Garcia-Santisteban et al., 2013) and, thus, both genes also could be involved in senescence."
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"Structural Comparisons of Activator-Bound USP12 and Ub-Conjugated USP46(A) Superposition of UAF1-bound USP12 and UAF1-WDR20-bound USP12 highlighting stabilization of BL1 and BL3 in the ternary complex."
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"As stated earlier, UAF1 interacts with two other USPs, USP12 and USP46."
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No evidence text available
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"3.2 The USP12 and UAF1 complex has 1:2 stoichiometry."
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"Overall, the UAF1, USP12, and WDR20 complex is distinguished from the two free USP12 structures and the two UAF1 and USP12 complex structures by the apparent stabilization of most flexible structural elements found in the DUB enzyme."
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"The human UAF1 or UAF1-USP12 complex was expressed and purified from Hi5 insect cells."
27373336
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No evidence text available
19075014
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"Despite a potential salt bridge and a small number of van der Waal interactions between the two beta-propellers, UAF1 binding to USP12 has little effect on the association of WDR20 with the enzyme (XREF_SUPPLEMENTARY)."
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"The human UAF1 or UAF1-USP12 complex was expressed and purified from Hi5 insect cells."
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"For example, USP1, USP12 and USP46 can interact with WD40-repeat protein USP1 associated factor 1 (UAF1), and UAF1 stimulates the catalytic activity of human USP1 through the formation of a tight complex [XREF_BIBR]."
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"In the free USP12 structure and in the F222 space group structure of the UAF1 and USP12 complex, this loop is excluded from the catalytic cleft by BL1 and BL2."
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"To confirm that these structural differences of USP12 are associated with UAF1 binding but not crystal packing or resolution limit, we crystallized the UAF1-USP12 complex in another space group (C2) and determined its structure at a 2.3 Å resolution."
27373336
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No evidence text available
19075014
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"Starting at the tip of the Fingers domain, the UAF1-USP12 interface directly stabilizes the distal region of the USP12 Pinky Finger ( Figure 3A ), which is either disordered or in a different conformation in the two USP12-alone structures."
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"In line with these observed dual binding states, we show here that USP12 and UAF1 complex has 1:2 stoichiometry in solution, with a two-step binding at 4nM and 325nM respectively."
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"Therefore, complete destabilization of BL1 per se is not the ultimate effect of UAF1 binding for USP12 activation."
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"Although BL1 is destabilized in the crystal structures of the UAF1-USP12 complex, it is expected to adopt a productive conformation upon substrate binding and play an important role in adjusting the topology of the Ub tail by fine-tuning its preceding region."
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"(C and D) A close-up comparison of the Pinky Finger, PK Helix, and PK Loop between free and UAF1-bound USP12 structures, with the two free USP12 structures aligned in (C)."
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"Binding of WDR20 to the UAF1-USP12 complex induces multiple structural changes in the enzyme, converting it into a compact form similar to Ub-bound USP46."
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"The predominant USP12-WDR20 interface also involves major structural elements that are topologically connected to UAF1 binding in the UAF1-USP12 structures."
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"In contrast, the PK Loop in the UAF1-USP12 complex structure appears to adopt a flexible structure, as evidenced by the absence of its electron density."
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No evidence text available
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"(E and F) A close-up comparison of the PK Loop and BL1 between free and UAF1-bound USP12 structures."
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"Asterisks indicate backbone groups that are part of the intermolecular hydrogen bond network. (H) Interactions of wild-type (WT) and mutant USP12 with UAF1 assessed by immunoprecipitation (IP) and western blot using an in vitro transcription and translation system.the UAF1-USP12 complex, in addition to the N-terminal WD40repeat domain and a central ancillary domain(Figures 2A, 2B, 2D, and S2A-S2E) Structural Differences between Free and UAF1-Bound USP12(A) Side view of the interface between UAF1 (orange) and the tip of USP12 Pinky Finger (green)."
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"Results confirmed the interaction between endogenous Usp12, Uaf-1 and WDR20 in PC cells."
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"Overall, the UAF1-USP12-WDR20 complex is distinguished from the two free USP12 structures and the two UAF1-USP12 complex structures by the apparent stabilization of most flexible structural elements found in the DUB."
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"Four basic UAF1 residues potentially responsible for recognizing the acidic tail of SLIM are shown in sticks. (E) Electrostatic surface potential of UAF1 SLD. (F) Ribbon diagram of the UAF1-USP12 interface viewed from the Thumb-Palm scaffold of the enzyme, with zinc shown as a yellow sphere."
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"The structurally regularized Pinky Finger in UAF1-bound USP12 is indicated in (D)."
27373336
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"Even though BL1 of USP12 is collapsed in our UAF1-USP12 complex structures, Ub binding is expected to stabilize it via substrate-induced fit, as seen in other USPs and the Ub-conjugated USP46 structures."
27373336
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"Despite a potential salt bridge and a small number of van der Waal interactions between the two β propellers, UAF1 binding to USP12 has little effect on the association of WDR20 with the enzyme (A and[MISSING/INVALID CREDENTIALS: limited to 200 char for Elsevier]"
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"In the two UAF1-USP12 complex structures, UAF1 binding appears to induce BL1 destabilization via a chain of conformational changes, whereas the WDR20-USP12 interaction seems to overwrite the allosteric effect of UAF1 by (D and E) Rates of Ub-AMC hydrolysis by wild-type (WT) and mutant USP12 in the presence of UAF1 (D) or WDR20 (E)."
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"Overall, the UAF1-USP12-WDR20 complex is distinguished from the two free USP12 structures and the two UAF1-USP12 complex structures by the apparent stabilization of most flexible structural elements found in the DUB."
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"A comparison between the activities of the UAF1-USP12 and those of the UAF1-USP12-WDR20 complex yielded a similar conclusion for WDR20, which also potentiates the enzyme by enhancing its k cat ."
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