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Ubiquitin binds USP2. 32 / 42
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"Consistent with this hypothesis, the structures of USP2, USP8, and OTUB2 with Ub bound in the distal binding site reveal that the relevant serine residues (Ser57 for USP2, XREF_FIG K; Ser20 and 57 for USP8, XREF_FIG J and 6M; and Ser65 for OTUB2, XREF_FIG N) are solvent exposed in the distal binding sites (XREF_FIG H)."
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"For USP16 we chose the model based on the USP2–Ub complex (PDB ID: ;), which has a sequence identity of 34%."
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"In addition, a layer of ordered water molecules mediates key interactions between ubiquitin and USP2."
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"The kinetic constants are similar to those determined for native monoubiquitin, suggesting that the conjugation of an AMC group to the C-terminus of ubiquitin does not affect the kinetic behavior of ubiquitin binding to USP2."
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"Kinetic studies with peptidic substrates and truncated ubiquitin mutants show that neither the C-terminal peptide nor ubiquitin deletion mutants lacking the C-terminal residues bind to USP2 with detectable affinity."
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"Notably, both native ubiquitin and Ub-AMC bind to USP2 with monophasic kinetics."
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"This suggests that ubiquitin binds to USP2 in a single step despite that USP2 catalytic core interacts with ubiquitin at multiple sites as revealed by the cocrystal structure of USP2-ubiquitin complex ( xref )."
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"These values are close to those determined for ubiquitin binding to the wild-type USP2 ( k on = 0.40 ± 0.01 μM −1 s −1 and k off = 2.64 ± 0.56 s −1 )."
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"Specific Interactions of USP2 with Ubiquitin."
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"However, for a significant fraction of the total contact area the interactions are indirect, as numerous solvent molecules mediate H bond interactions between ubiquitin backbone and USP2 side chain/backbone atoms ( xref D and 2E)."
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"After having shown that ubiquitin interacts with USP2 by using two sites, we tried to determine the individual contribution of the isolated interactions to the ubiquitin binding affinity."
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"Recombinant full-length human USP5/ISOT (residues 1–835) was purified from bacteria, inhibited with a ubiquitin-based suicide substrate and crystallized at 20 °C. A 2.8 Å resolution dataset was collected yielding a structure after phases were obtained by molecular replacement (covalent ubiquitin-USP2 complex (PDB code 2IBI) and the USP5 ZnF-UBP domain (PDB code 2G43)) with good crystallographic statistics ( xref )."
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"This shows that USP2 binds ubiquitin with significant affinity, and it suggests that product inhibition by ubiquitin is a general feature of the USP family of proteases."
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"In summary, our data show that the ubiquitin core and the ubiquitin C terminus alone bind USP2 very weakly with affinities at best in the high micromolar range, while full-length ubiquitin inhibits USP2 with a K i of 2.8 μM."
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"In the USP2 and ubiquitin complex the Finger domain is displaced outward to adopt the conformation observed in the other USP domains."
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"The control of Imd homeostasis by USP2 is associated with the hydrolysis of Imd linked K48-ubiquitin chains and the synergistic binding of USP2 and Imd to the proteasome, as evidenced by both mass-spectrometry analysis of USP2 partners and by co-immunoprecipitation experiments."
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"When ubiquitin binds to USP2, its core is recognized by the cupped hand."
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"Yet, such an inhibition will only be observed if the concentration of free ubiquitin is in the same range as the K for the interaction between ubiquitin and the protease.The structure of the USP2/ubiquitin complex shows that ubiquitin uses two interaction sites: its core (residues 1–71) binds into the cupped hand interacting with fingers and palm, and its C terminus (residues 72–76) binds into the substrate cleft at the catalytic center."
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"This is surprising, as, in the USP2/ubiquitin complex structure, the Gly75-Gly76 dipeptide binds into a narrow channel formed by the highly conserved residue Gln293 and is engaged in five potential H bond interactions."
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"USP2•Ub-AMC is the Michaelis complex of USP2 and Ub-AMC; USP2-Ub is an acyl-enzyme intermediate where ubiquitin is covalently bound to the USP2 active site cysteine through a thioester bond."
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"USP2•Ub is USP2 bound with ubiquitin following the deacylation reaction."
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"The USP2-ubiquitin interface in the area of the ubiquitin core ( 1–71 ) appears to be less hydrophobic than protease-inhibitor interfaces in general."
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"As observed for HAUSP xref and USP2 xref interactions with ubiquitin, a number of water molecules line the protein-protein interface including a loop of four ubiquitin residues (A46-Q49) that is involved in a number of polar interactions with the palm of PLpro ( xref )."
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"For USP16 we chose the model based on the USP2–Ub complex (PDB ID: ; 2HD5), which has a sequence identity of 34%."
32110315
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"Despite the addition of the fluorophore AMC to the C-terminus of ubiquitin, the binding kinetics of Ub-AMC to an inactive USP2 is similar to that of the native ubiquitin binding to the wild-type USP2 ( xref ), indicating that the AMC group does not disrupt the interaction of ubiquitin with the USP2 catalytic core."
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"Surprisingly, a single replacement of Ub residue 72 with its equivalent NEDD8 residue did not result in any inhibition (Fig.  xref , lane 13), suggesting that binding of the C-terminus of Ub to USP2 was not adequately stable."
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"This model also concluded that USP2 bound with the N-terminus of Ub to form a stable interaction and then bound to the C-terminus of Ub to ensure substrate specificity."
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"Modeling of ubiquitin into the central binding cavity by superposition of the USP2–ubiquitin complex structure (PDB entry( xref )) reveals that additional conformational changes in the finger region, including the zinc finger ribbon linking the two subdomains D1 and D2, are required to accommodate the distal ubiquitin core and avoid steric clashes ( xref D )."
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"Ubiquitin was used for the USP2/ubiquitin complex, while ubiquitin-aldehyde was used for the formation of corresponding USP7 complex."
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"Instead, no covalent bond is formed in the USP2/ubiquitin complex."
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"In the USP2/ubiquitin complex, Nζ of Lys407 occupies the same position as a solvent molecule in the USP7/ubiquitin-aldehyde complex.The principles of protein-protein interactions and the role of water molecules in molecular recognition have been studied by various groups (Janin, 1997, Jones and Thornton, 1996, Larsen et al., 1998)."
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"The USP2/ubiquitin complex was formed by mixing 40 μl USP2 (11.8 mg/ml in 15 mM potassium phosphate [pH 6.0], 100 mM NaCl, 25 mM DTT) and 20 μl ubiquitin (Sigma U6253, 10 mg/ml in H O demin) corresponding to a 1:2 molar ratio."
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