Structure of the HAUSP N-Terminal TRAF-Like Domain

<div><p>(A) Structure of the HAUSP TRAF-like domain in a ribbon diagram (left) and a surface representation (right). Secondary structural elements (left) and the putative substrate-binding groove (right) are labeled.</p> <p>(B) Sequence alignment of the HAUSP TRAF-like domain with other TRAF family members. Conserved residues are shown in yellow. Residues that interact with p53 through hydrogen bonds and van der Waals contacts are identified by green arrow heads and green squares, respectively. Residues that interact with MDM2 through hydrogen bonds and van der Waals contacts are indicated by red arrow heads and red squares, respectively. Conserved residues that are involved in binding to peptides in other TRAF family proteins, but not in HAUSP, are colored red and indicated by purple background.</p></div

Structural Comparison of Peptide Binding by HAUSP Reveals a Consensus Sequence

<div><p>(A) MDM2 peptide (red) binds to the same surface groove as the p53 peptide (magenta). Residues from MDM2 and p53 are shown in yellow and green, respectively.</p> <p>(B) Superposition of three HAUSP-binding peptides derived from MDM2 (red), p53 (magenta), and EBNA1 (green). The HAUSP TRAF-like domain is shown in a transparent surface representation, with critical residues shown in brown.</p> <p>(C) Structural alignment of HAUSP-binding peptides reveals a consensus sequence. The HAUSP surface groove (in a transparent surface representation) for binding to the consensus tetrapeptide is shown in the left panel. The consensus sequence is shown in the right panel.</p></div

Structural Basis of MDM2 Recognition by HAUSP

<div><p>(A) Overall structure of the HAUSP TRAF-like domain bound to MDM2 peptide is shown in a ribbon diagram (left) and in a surface representation (right). The important MDM2 residues are highlighted in yellow.</p> <p>(B) A stereo view of the specific interactions between MDM2 and HAUSP. These interactions are more extensive than those between p53 and HAUSP. Hydrogen bonds are represented by red dashed lines. All interacting residues are labeled.</p></div

Structure of an Extended HAUSP Fragment

<div><p>(A) Structure of a HAUSP fragment (residues 53–560) that contains both the substrate-binding (green) and the catalytic domains. Binding sites for ubiquitin and substrate are indicated. The linker sequences between these two domains have high-temperature factors and are flexible in the crystals.</p> <p>(B) A structure-based model showing HAUSP bound to an ubiquitylated MDM2. Only one ubiquitin moiety and the MDM2 peptide are shown in this model.</p></div

HAUSP Preferentially Forms a Stable HAUSP–MDM2 Complex in the Presence of Excess p53

<div><p>(A) The TRAF-like domain of HAUSP is responsible for binding to MDM2. Various HAUSP fragments were individually incubated with MDM2 protein (residues 170–423) and their interactions were examined by gel filtration. The results are summarized here.</p> <p>(B) Identification of a minimal HAUSP-binding element in MDM2. Various MDM2 fragments were individually incubated with HAUSP TRAF-like domain (residues 53–206) and their interactions were examined by gel filtration. The results are summarized here.</p> <p>(C) HAUSP preferentially forms a stable HAUSP–MDM2 complex in the presence of excess p53. HAUSP (residues 1–206) interacts with both p53 (residues 351–382, upper panel) and MDM2 (residues 208–289, middle panel). However, in the presence of a 10-fold excess amount of p53, HAUSP formed a stable complex only with MDM2 (lower panel). The relevant peak fractions were visualized by SDS-PAGE followed by Coomassie staining.</p> <p>(D) Determination of binding affinities between the HAUSP TRAF-like domain (residues 53–206) and peptides derived from p53 and MDM2 by ITC. The p53 and MDM2 peptides contain residues 351–382 and 208–242, respectively. The binding affinities for the p53 and MDM2 peptides are 3 and 21 μM, respectively.</p></div

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<p>Dibenzothiophene (DBT) and their derivatives, accounting for the major part of the sulfur components in crude oil, make one of the most significant pollution sources. The DBT sulfone monooxygenase BdsA, one of the key enzymes in the “4S” desulfurization pathway, catalyzes the oxidation of DBT sulfone to 2′-hydroxybiphenyl 2-sulfonic acid (HBPSi). Here, we determined the crystal structure of BdsA from Bacillus subtilis WU-S2B, at the resolution of 2.2 Å, and the structure of the BdsA-FMN complex at 2.4 Å. BdsA and the BdsA-FMN complex exist as tetramers. DBT sulfone was placed into the active site by molecular docking. Seven residues (Phe12, His20, Phe56, Phe246, Val248, His316, and Val372) are found to be involved in the binding of DBT sulfone. The importance of these residues is supported by the study of the catalytic activity of the active site variants. Structural analysis and enzyme activity assay confirmed the importance of the right position and orientation of FMN and DBT sulfone, as well as the involvement of Ser139 as a nucleophile in catalysis. This work combined with our previous structure of DszC provides a systematic structural basis for the development of engineered desulfurization enzymes with higher efficiency and stability.</p

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<p>Dibenzothiophene (DBT) and their derivatives, accounting for the major part of the sulfur components in crude oil, make one of the most significant pollution sources. The DBT sulfone monooxygenase BdsA, one of the key enzymes in the “4S” desulfurization pathway, catalyzes the oxidation of DBT sulfone to 2′-hydroxybiphenyl 2-sulfonic acid (HBPSi). Here, we determined the crystal structure of BdsA from Bacillus subtilis WU-S2B, at the resolution of 2.2 Å, and the structure of the BdsA-FMN complex at 2.4 Å. BdsA and the BdsA-FMN complex exist as tetramers. DBT sulfone was placed into the active site by molecular docking. Seven residues (Phe12, His20, Phe56, Phe246, Val248, His316, and Val372) are found to be involved in the binding of DBT sulfone. The importance of these residues is supported by the study of the catalytic activity of the active site variants. Structural analysis and enzyme activity assay confirmed the importance of the right position and orientation of FMN and DBT sulfone, as well as the involvement of Ser139 as a nucleophile in catalysis. This work combined with our previous structure of DszC provides a systematic structural basis for the development of engineered desulfurization enzymes with higher efficiency and stability.</p

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<p>Dibenzothiophene (DBT) and their derivatives, accounting for the major part of the sulfur components in crude oil, make one of the most significant pollution sources. The DBT sulfone monooxygenase BdsA, one of the key enzymes in the “4S” desulfurization pathway, catalyzes the oxidation of DBT sulfone to 2′-hydroxybiphenyl 2-sulfonic acid (HBPSi). Here, we determined the crystal structure of BdsA from Bacillus subtilis WU-S2B, at the resolution of 2.2 Å, and the structure of the BdsA-FMN complex at 2.4 Å. BdsA and the BdsA-FMN complex exist as tetramers. DBT sulfone was placed into the active site by molecular docking. Seven residues (Phe12, His20, Phe56, Phe246, Val248, His316, and Val372) are found to be involved in the binding of DBT sulfone. The importance of these residues is supported by the study of the catalytic activity of the active site variants. Structural analysis and enzyme activity assay confirmed the importance of the right position and orientation of FMN and DBT sulfone, as well as the involvement of Ser139 as a nucleophile in catalysis. This work combined with our previous structure of DszC provides a systematic structural basis for the development of engineered desulfurization enzymes with higher efficiency and stability.</p

Measurement of binding affinity between LidA and 9 kinds of Rabs by ITC.

<p>(A–G) Raw ITC data. Top panel: twenty injections of Rab2 (A), Rab4 (B), Rab6 (C), Rab7 (D), Rab9 (E), Rab11 (F), Rab20 (G) solutions were titrated into LidA(188-580) solution in ITC cell. The area of each injection peak corresponds to the total heat released for that injection. Bottom panel: the binding isotherm for these Rabs and LidA(188-580) interaction, the integrated heat is plotted against the stoichiometry of 1∶1, data fitting revealed a binding affinity as shown. (H) Raw ITC data. Top panel: twenty injections of Rab22 (H) solutions were titrated into LidA(FL) solution ITC cell, the experiment conditions was exactly the same as the top one unless the LidA is full-length. The <i>K_D</i> values of these Rabs and LidA are shown.</p