(1)H, (13)C and (15)N backbone resonance assignment for the 40.5 kDa catalytic domain of Ubiquitin Specific Protease 7 (USP7).

The deubiquitinase Ubiquitin Specific Protease 7 (USP7) is part of the regulatory cascade of proteins that modulates the activity of the tumor suppressor protein p53. Deubiquitination of its target Murine Double Minute 2 (MDM2) leads to increased proteosomal degradation of p53. Consequently, USP7 has emerged as an attractive oncology target because its inhibition stabilizes p53, thereby promoting p53-dependent apoptosis in cancer cells. Here we report the backbone resonance assignment for the 40.5 kDa catalytic domain of USP7.

Small-molecule ligands bind to a distinct pocket in Ras and inhibit SOS-mediated nucleotide exchange activity.

The Ras gene is frequently mutated in cancer, and mutant Ras drives tumorigenesis. Although Ras is a central oncogene, small molecules that bind to Ras in a well-defined manner and exert inhibitory effects have not been uncovered to date. Through an NMR-based fragment screen, we identified a group of small molecules that all bind to a common site on Ras. High-resolution cocrystal structures delineated a unique ligand-binding pocket on the Ras protein that is adjacent to the switch I/II regions and can be expanded upon compound binding. Structure analysis predicts that compound-binding interferes with the Ras/SOS interactions. Indeed, selected compounds inhibit SOS-mediated nucleotide exchange and prevent Ras activation by blocking the formation of intermediates of the exchange reaction. The discovery of a small-molecule binding pocket on Ras with functional significance provides a new direction in the search of therapeutically effective inhibitors of the Ras oncoprotein.

The structure of the extracellular domain of the jumping translocation breakpoint protein reveals a variation of the midkine fold.

Jumping Translocation Breakpoint (JTB) is an orphan receptor that is conserved from nematodes to humans and whose gene expression in humans is strikingly upregulated in diverse types of cancers. Translocations occur frequently at the hJTB genomic locus, leading to multiple copies of a truncated JTB gene, which potentially encodes a soluble secreted ectodomain. In addition, JTB and its orthologs likely represent a unique and ancient protein family since homologs could not be identified by direct sequence comparison. In the present study, we have determined the NMR solution structure of the N-terminal ectodomain of human JTB, showing that its fold architecture is a new variant of a three-ß-strand antiparallel ß-meander. The JTB structure has a distant relationship to the midkine/pleiotrophin fold, particularly in the conservation of distinctive disulfide bridge patterns. The structure of this newly characterized small cysteine-rich domain suggests potential involvement of JTB in interactions with proteins or extracellular matrix and may help to uncover the elusive biological functions of this protein.

Modulation of K11-linkage formation by variable loop residues within UbcH5A.

Ubiquitination refers to the covalent addition of ubiquitin (Ub) to substrate proteins or other Ub molecules via the sequential action of three enzymes (E1, E2, and E3). Recent advances in mass spectrometry proteomics have made it possible to identify and quantify Ub linkages in biochemical and cellular systems. We used these tools to probe the mechanisms controlling linkage specificity for UbcH5A. UbcH5A is a promiscuous E2 enzyme with an innate preference for forming polyubiquitin chains through lysine 11 (K11), lysine 48 (K48), and lysine 63 (K63) of Ub. We present the crystal structure of a noncovalent complex between Ub and UbcH5A. This structure reveals an interaction between the Ub surface flanking K11 and residues adjacent to the E2 catalytic cysteine and suggests a possible role for this surface in formation of K11 linkages. Structure-guided mutagenesis, in vitro ubiquitination and quantitative mass spectrometry have been used to characterize the ability of residues in the vicinity of the E2 active site to direct synthesis of K11- and K63-linked polyubiquitin. Mutation of critical residues in the interface modulated the linkage specificity of UbcH5A, resulting in generation of more K63-linked chains at the expense of K11-linkage synthesis. This study provides direct evidence that the linkage specificity of E2 enzymes may be altered through active-site mutagenesis.

Ubiquitin binding to A20 ZnF4 is required for modulation of NF-κB signaling.

Inactivating mutations in the ubiquitin (Ub) editing protein A20 promote persistent nuclear factor (NF)-κB signaling and are genetically linked to inflammatory diseases and hematologic cancers. A20 tightly regulates NF-κB signaling by acting as an Ub editor, removing K63-linked Ub chains and mediating addition of Ub chains that target substrates for degradation. However, a precise molecular understanding of how A20 modulates this pathway remains elusive. Here, using structural analysis, domain mapping, and functional assays, we show that A20 zinc finger 4 (ZnF4) does not directly interact with E2 enzymes but instead can bind mono-Ub and K63-linked poly-Ub. Mutations to the A20 ZnF4 Ub-binding surface result in decreased A20-mediated ubiquitination and impaired regulation of NF-κB signaling. Collectively, our studies illuminate the mechanistically distinct but biologically interdependent activities of the A20 ZnF and ovarian tumor (OTU) domains that are inherent to the Ub editing process and, ultimately, to regulation of NF-κB signaling.

Structural and functional analysis of the PDZ domains of human HtrA1 and HtrA3.

High-temperature requirement A (HtrA) and its homologs contain a serine protease domain followed by one or two PDZ domains. Bacterial HtrA proteins and the mitochondrial protein HtrA2/Omi maintain cell function by acting as both molecular chaperones and proteases to manage misfolded proteins. The biological roles of the mammalian family members HtrA1 and HtrA3 are less clear. We report a detailed structural and functional analysis of the PDZ domains of human HtrA1 and HtrA3 using peptide libraries and affinity assays to define specificity, structural studies to view the molecular details of ligand recognition, and alanine scanning mutagenesis to investigate the energetic contributions of individual residues to ligand binding. In common with HtrA2/Omi, we show that the PDZ domains of HtrA1 and HtrA3 recognize hydrophobic polypeptides, and while C-terminal sequences are preferred, internal sequences are also recognized. However, the details of the interactions differ, as different domains rely on interactions with different residues within the ligand to achieve high affinity binding. The results suggest that mammalian HtrA PDZ domains interact with a broad range of hydrophobic binding partners. This promiscuous specificity resembles that of bacterial HtrA family members and suggests a similar function for recognizing misfolded polypeptides with exposed hydrophobic sequences. Our results support a common activation mechanism for the HtrA family, whereby hydrophobic peptides bind to the PDZ domain and induce conformational changes that activate the protease. Such a mechanism is well suited to proteases evolved for the recognition and degradation of misfolded proteins.

Structure of SAP18: a ubiquitin fold in histone deacetylase complex assembly.

Signal transduction pathways are frequently found to repress transcription of target genes in the absence of stimulation and, conversely, to upregulate transcription in the presence of a signal. Transcription factors are central in this dual regulatory mechanism and widely use a generalized mechanism to repress transcription through recruitment of a Sin3-histone deacetylase (HDAC) complex to their binding sites on DNA. The protein SAP18 (Sin3-associated polypeptide of 18 kDa) has been shown to play a key role in gene-specific recruitment of the HDAC complex by a number of transcription factors including Gli, GAGA, and Bicoid. The solution structure of SAP18 reveals a ubiquitin-like fold with several large loop insertions relative to other family members. This fold supports the functional role of SAP18 as a protein-protein adapter module and provides insight for how SAP18 may bridge the Sin3-HDAC complex to transcription factors.

Structures of APRIL-receptor complexes: like BCMA, TACI employs only a single cysteine-rich domain for high affinity ligand binding.

TACI is a member of the tumor necrosis factor receptor superfamily and serves as a key regulator of B cell function. TACI binds two ligands, APRIL and BAFF, with high affinity and contains two cysteine-rich domains (CRDs) in its extracellular region; in contrast, BCMA and BR3, the other known high affinity receptors for APRIL and BAFF, respectively, contain only a single or partial CRD. However, another form of TACI exists wherein the N-terminal CRD is removed by alternative splicing. We find that this shorter form is capable of ligand-induced cell signaling and that the second CRD alone (TACI_d2) contains full affinity for both ligands. Furthermore, we report the solution structure and alanine-scanning mutagenesis of TACI_d2 along with co-crystal structures of APRIL.TACI_d2 and APRIL.BCMA complexes that together reveal the mechanism by which TACI engages high affinity ligand binding through a single CRD, and we highlight sources of ligand-receptor specificity within the APRIL/BAFF system.

BAFF/BLyS receptor 3 comprises a minimal TNF receptor-like module that encodes a highly focused ligand-binding site.

BAFF/BLyS, a member of the tumor necrosis family (TNF) superfamily of ligands, is a crucial survival factor for B cells. BAFF binds three receptors, TACI, BCMA, and BR3, with signaling through BR3 being essential for promoting B cell function. Typical TNF receptor (TNFR) family members bind their cognate ligands through interactions with two cysteine-rich domains (CRDs). However, the extracellular domain (ECD) of BR3 consists of only a partial CRD, with cysteine spacing distinct from other modules described previously. Herein, we report the solution structure of the BR3 ECD. A core region of only 19 residues adopts a stable structure in solution. The BR3 fold is analogous to the first half of a canonical TNFR CRD but is stabilized by an additional noncanonical disulfide bond. BAFF-binding determinants were identified by shotgun alanine-scanning mutagenesis of the BR3 ECD expressed on phage. Several of the key BAFF-binding residues are presented from a beta-turn that we have shown previously to be sufficient for ligand binding when transferred to a structured beta-hairpin scaffold [Kayagaki, N., Yan, M., Seshasayee, D., Wang, H., Lee, W., French, D. M., Grewal, I. S., Cochran, A. G., Gordon, N. C., Yin, J., Starovasnik, M. A, and Dixit, V. M. (2002) Immunity 10, 515-524]. Outside of the turn, mutagenesis identifies additional hydrophobic contacts that enhance the BAFF-BR3 interaction. The crystal structure of the minimal hairpin peptide, bhpBR3, in complex with BAFF reveals intimate packing of the six-residue BR3 turn into a cavity on the ligand surface. Thus, BR3 binds BAFF through a highly focused interaction site, unprecedented in the TNFR family.

Solution structure of a phage-derived peptide antagonist in complex with vascular endothelial growth factor.

Vascular endothelial growth factor (VEGF) is a potent endothelial cell-specific mediator of angiogenesis and vasculogenesis. VEGF is involved pathologically in cancer, proliferative retinopathy and rheumatoid arthritis, and as such represents an important therapeutic target. Three classes of disulfide-constrained peptides that antagonize binding of the VEGF dimer to its receptors, KDR and Flt-1, were identified previously using phage display methods. NMR studies of a representative peptide from the most potent class of these peptide antagonists, v107 (GGNECDAIRMWEWECFERL), were undertaken to characterize its interactions with VEGF. v107 has no defined structure free in solution, but binding to VEGF induces folding of the peptide. The solution structure of the VEGF receptor-binding domain-v107 complex was determined using 3940 (1970 per VEGF monomer) internuclear distance and 476 (238 per VEGF monomer) dihedral angle restraints derived from NMR data obtained using samples containing either (13)C/(15)N-labeled protein plus excess unlabeled peptide or (13)C/(15)N-labeled peptide plus excess unlabeled protein. Residual dipolar coupling restraints supplemented the structure determination of the complex and were found to increase significantly both the global precision of VEGF in the complex and the agreement with available crystal structures of VEGF. The calculated ensemble of structures is of high precision and is in excellent agreement with the experimental restraints. v107 has a turn-helix conformation with hydrophobic residues partitioned to one face of the peptide and polar or charged residues at the other face. Contacts between two v107 peptides and the VEGF dimer are mediated by primarily hydrophobic side-chain interactions. The v107-binding site on VEGF overlaps partially with the binding site of KDR and is similar to that for domain 2 of Flt-1. The structure of the VEGF-v107 complex provides new insight into how binding to VEGF can be achieved that may be useful for the design of small molecule antagonists.