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

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

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

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

Engineered Lignin in Poplar Biomass Facilitates Cu-Catalyzed Alkaline-Oxidative Pretreatment

Both untransformed poplar and genetically modified “zip-lignin” poplar, in which additional ester bonds were introduced into the lignin backbone, were subjected to mild alkaline and copper-catalyzed alkaline hydrogen peroxide (Cu-AHP) pretreatment. Our hypothesis was that the lignin in zip-lignin poplar would be removed more easily than lignin in untransformed poplar during this alkaline pretreatment, resulting in higher sugar yields following enzymatic hydrolysis. We observed improved glucose and xylose hydrolysis yields for zip-lignin poplar compared to untransformed poplar following both alkaline-only pretreatment (56% glucose yield for untransformed poplar compared to 67% for zip-lignin poplar) and Cu-AHP pretreatment (77% glucose yield for untransformed poplar compared to 85% for zip-lignin poplar). Compositional analysis, glycome profiling, and microscopy all supported the notion that the ester linkages increase delignification and improve sugar yields. Essentially no differences were noted in the molecular weight distributions of solubilized lignins between the zip-lignin poplar and the control line. Significantly, when zip-lignin poplar was utilized as the feedstock, hydrogen peroxide, catalyst, and enzyme loadings could all be substantially reduced while maintaining high sugar yields