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The Fanconi Anemia (FA) DNA repair pathway is essential for the recognition and repair of DNA interstrand crosslinks (ICL). Inefficient repair of these ICL can lead to leukemia and bone marrow failure. A critical step in the pathway is the monoubiquitination of FANCD2 by the RING E3 ligase FANCL. FANCL comprises 3 domains, a RING domain that interacts with E2 conjugating enzymes, a central domain required for substrate interaction, and an N-terminal E2-like fold (ELF) domain. The ELF domain is found in all FANCL homologues, yet the function of the domain remains unknown. We report here that the ELF domain of FANCL is required to mediate a non-covalent interaction between FANCL and ubiquitin. The interaction involves the canonical Ile44 patch on ubiquitin, and a functionally conserved patch on FANCL. We show that the interaction is not necessary for the recognition of the core complex, it does not enhance the interaction between FANCL and Ube2T, and is not required for FANCD2 monoubiquitination in vitro. However, we demonstrate that the ELF domain is required to promote efficient DNA damage-induced FANCD2 monoubiquitination in vertebrate cells, suggesting an important function of ubiquitin binding by FANCL in vivo., (© 2015 by The American Society for Biochemistry and Molecular Biology, Inc.)

Parkin is an E3-ubiquitin ligase belonging to the RBR (RING-InBetweenRING-RING family), and is involved in the neurodegenerative disorder Parkinson's disease. Autosomal recessive juvenile Parkinsonism, which is one of the most common familial forms of the disease, is directly linked to mutations in the parkin gene. However, the molecular mechanisms of Parkin dysfunction in the disease state remain to be established. We now demonstrate that the ubiquitin-like domain of Parkin functions to inhibit its autoubiquitination. Moreover pathogenic Parkin mutations disrupt this autoinhibition, resulting in a constitutively active molecule. In addition, we show that the mechanism of autoregulation involves ubiquitin binding by a C-terminal region of Parkin. Our observations provide important molecular insights into the underlying basis of Parkinson's disease, and in the regulation of RBR E3-ligase activity.

Purified preparations of the recombinant b'x domain fragment of human protein-disulphide isomerase (PDI), which are homogeneous by mass spectrometry and sodium dodecyl sulfate polyacrylamide gel electrophoresis, comprise more than one species when analyzed by ion-exchange chromatography and nondenaturing polyacrylamide gel electrophoresis. These species were resolved and shown to be monomer and dimer by analytical ultracentrifugation and analytical size-exclusion chromatography. Spectroscopic properties indicate that the monomeric species corresponds to the "capped" conformation observed in the x-ray structure of the I272A mutant of b'x (Nguyen, Wallis, Howard, Haapalainen, Salo, Saaranen, Sidhu, Wierenga, Freedman, Ruddock, and Williamson, J Mol Biol 2008;383:1144-1155) in which the x region binds to a hydrophobic patch on the surface of the b' domain; conversely, the dimeric species has an "open" or "uncapped" conformation in which the x region does not bind to this surface. The larger bb'x fragment of human PDI shows very similar behavior to b'x and can be resolved into a capped monomeric species and an uncapped dimer. Preparations of recombinant b' domain of human PDI and of the bb' domain pair are found exclusively as dimers. Full-length PDI is known to comprise a mixture of monomeric and dimeric species, whereas the isolated a, b, and a' domains of PDI are found exclusively as monomers. These results show that the b' domain of human PDI tends to form homodimers--both in isolation and in other contexts--and that this tendency is moderated by the adjacent x region, which can bind to a surface patch on the b' domain.

PDI (protein disulfide-isomerase) catalyses the formation of native disulfide bonds of secretory proteins in the endoplasmic reticulum. PDI consists of four thioredoxin-like domains, of which two contain redox-active catalytic sites (a and a'), and two do not (b and b'). The b' domain is primarily responsible for substrate binding, although the nature and specificity of the substrate-binding site is still poorly understood. In the present study, we show that the b' domain of human PDI is in conformational exchange, but that its structure is stabilized by the addition of peptide ligands or by binding the x-linker region. The location of the ligand-binding site in b' was mapped by NMR chemical shift perturbation and found to consist primarily of residues from the core beta-sheet and alpha-helices 1 and 3. This site is where the x-linker region binds in the X-ray structure of b'x and we show that peptide ligands can compete with x binding at this site. The finding that x binds in the principal ligand-binding site of b' further supports the hypothesis that x functions to gate access to this site and so modulates PDI activity.

Protein-disulfide isomerase (PDI), a critical enzyme responsible for oxidative protein folding in the eukaryotic endoplasmic reticulum, is composed of four thioredoxin domains a, b, b', a', and a linker x between b' and a'. Ero1-Lalpha, an oxidase for human PDI (hPDI), has been determined to have one molecular flavin adenine dinucleotide (FAD) as its prosthetic group. Oxygen consumption assays with purified recombinant Ero1-Lalpha revealed that it utilizes oxygen as a terminal electron acceptor producing one disulfide bond and one molecule of hydrogen peroxide per dioxygen molecule consumed. Exogenous FAD is not required for recombinant Ero1-Lalpha activity. By monitoring the reactivation of denatured and reduced RNase A, we reconstituted the Ero1-Lalpha/hPDI oxidative folding system in vitro and determined the enzymatic activities of hPDI in this system. Mutagenesis studies suggested that the a' domain of hPDI is much more active than the a domain in Ero1-Lalpha-mediated oxidative folding. A domain swapping study revealed that one catalytic thioredoxin domain to the C-terminal of bb'x, whether a or a', is essential in Ero1-Lalpha-mediated oxidative folding. These data, combined with a pull-down assay and isothermal titration calorimetry measurements, enabled the minimal element for binding with Ero1-Lalpha to be mapped to the b'xa' fragment of hPDI.

Protein disulphide isomerase (PDI) is a key multi-domain protein folding catalyst in the endoplasmic reticulum. The b' domain of PDI is essential for the non-covalent binding of incompletely folded protein substrates. Earlier, we defined the substrate binding site in the b' domain of human PDI by modelling and mutagenesis studies. Here, we show by fluorescence and NMR that recombinant human PDI b'x (comprising the b' domain and the subsequent x linker region) can assume at least two different conformations in solution. We have screened mutants in the b'x region to identify mutations that favour one of these conformers in recombinant b'x, and isolated and characterised examples of both types. We have crystallised one mutant of b'x (I272A mutation) in which one conformer is stabilized, and determined its crystal structure to a resolution of 2.2 A. This structure shows that the b' domain has the typical thioredoxin fold and that the x region can interact with the b' domain by "capping" a hydrophobic site on the b' domain. This site is most likely the substrate binding site and hence such capping will inhibit substrate binding. All of the mutations we previously reported to inhibit substrate binding shift the equilibrium towards the capped conformer. Hence, these mutations act by altering the natural equilibrium and decreasing the accessibility of the substrate binding site. Furthermore, we have confirmed that the corresponding structural transition occurs in the wild type full-length PDI. A cross-comparison of our data with that for other PDI-family members, Pdi1p and ERp44, suggests that the x region of PDI can adopt alternative conformations during the functional cycle of PDI action and that these are linked to the ability of PDI to interact with folding substrates.

Signal peptide identification is of immense importance in drug design. Accurate identification of signal peptides is the first critical step to be able to change the direction of the targeting proteins and use the designed drug to target a specific organelle to correct a defect. Because experimental identification is the most accurate method, but is expensive and time-consuming, an efficient and affordable automated system is of great interest. In this article, we propose using an adapted neural network, called a bio-basis function neural network, and decision trees for predicting signal peptides. The bio-basis function neural network model and decision trees achieved 97.16% and 97.63% accuracy respectively, demonstrating that the methods work well for the prediction of signal peptides. Moreover, decision trees revealed that position P(1'), which is important in forming signal peptides, most commonly comprises either leucine or alanine. This concurs with the (P(3)-P(1)-P(1')) coupling model.