NORMAL GENOMIC

• Kurz T et al.
• Dcn1 functions as a scaffold-type E3 ligase for cullin neddylation.
• Mol Cell. 2008; 29: 23-35
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• Cullin-based E3 ubiquitin ligases are activated through modification ofthe cullin subunit with the ubiquitin-like protein Nedd8. Dcn1 regulatescullin neddylation and thus ubiquitin ligase activity. Here we describethe 1.9 A X-ray crystal structure of yeast Dcn1 encompassing an N-terminalubiquitin-binding (UBA) domain and a C-terminal domain of uniquearchitecture, which we termed PONY domain. A conserved surface on Dcn1 isrequired for direct binding to cullins and for neddylation. The reciprocalbinding site for Dcn1 on Cdc53 is located approximately 18 A from the siteof neddylation. Dcn1 does not require cysteine residues for catalyticfunction, and directly interacts with the Nedd8 E2 Ubc12 on a surface thatoverlaps with the E1-binding site. We show that Dcn1 is necessary andsufficient for cullin neddylation in a purified recombinant system. Takentogether, these data demonstrate that Dcn1 is a scaffold-like E3 ligasefor cullin neddylation.

• Trempe JF et al.
• Mechanism of Lys48-linked polyubiquitin chain recognition by the Mud1 UBAdomain.
• EMBO J. 2005; 24: 3178-89
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• The ubiquitin-pathway associated (UBA) domain is a 40-residuepolyubiquitin-binding motif. The Schizosaccharomyces pombe protein Mud1 isan ortholog of the Saccharomyces cerevisiae DNA-damage response proteinDdi1 and binds to K48-linked polyubiquitin through its UBA domain. We havesolved the crystal structure of Mud1 UBA at 1.8 angstroms resolution,revealing a canonical three-helical UBA fold. We have probed theinteractions of this domain using mutagenesis, surface plasmon resonance,NMR and analytical ultracentrifugation. We show that the ubiquitin-bindingsurface of Mud1 UBA extends beyond previously recognized motifs and can befunctionally dissected into primary and secondary ubiquitin-binding sites.Mutation of Phe330 to alanine, a residue exposed between helices 2 and 3,significantly reduces the affinity of the Mud1 UBA domain for K48-linkedpolyubiquitin, despite leaving the primary binding surface functionallyintact. Moreover, K48-linked diubiquitin binds a single Mud1 UBA domaineven in the presence of excess UBA. We therefore propose a mechanism forthe recognition of K48-linked polyubiquitin chains by Mud1 in whichdiubiquitin units are specifically recognized by a single UBA domain.

• Kofler M, Motzny K, Beyermann M, Freund C
• Novel interaction partners of the CD2BP2-GYF domain.
• J Biol Chem. 2005; 280: 33397-402
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• The GYF domain of CD2BP2 serves as an adapter that recognizes proline-richsequences in intracellular proteins. Although the T cell adhesion moleculeCD2 and the core splicing protein SmB/B' were previously shown to interactwith CD2BP2-GYF, we are now using a general approach to identify putativeGYF domain target sites within the human proteome. The phagedisplay-derived recognition motif for CD2BP2-GYF is PPG(W/F/Y/M/L). SPOTanalysis confirmed that the GYF domain interacts with peptides from humanproteins containing the consensus site. Epitope mapping by NMRspectroscopy performed for several peptides revealed a conserved bindingsurface. A direct interaction of the CD2BP2-GYF domain with the novelprotein interaction partners PI31 and NPWBP was verified by yeasttwo-hybrid analysis.

• Ohno A et al.
• Structure of the UBA domain of Dsk2p in complex with ubiquitin moleculardeterminants for ubiquitin recognition.
• Structure. 2005; 13: 521-32
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• The ubiquitin-associated (UBA) domain is one of the most frequentlyoccurring motifs that recognize ubiquitin tags. Dsk2p, a UBA-containingprotein from Saccharomyces cerevisiae, is involved in theubiquitin-proteasome proteolytic pathway and has been implicated inspindle pole duplication. Here we present the solution structure of theUBA domain of Dsk2p (Dsk2(UBA)) in complex with ubiquitin. The structurereveals that the UBA domain uses a mode of ubiquitin recognition that issimilar to that of the CUE domain, another ubiquitin binding motif thatshares low sequence homology but high structural similarity with UBAdomains. These two domains, as well as the structurally unrelatedubiquitin binding motif UIM, provide a common, crucial recognition sitefor ubiquitin, comprising a hydrogen-bonding acceptor for the amide groupof Gly-47, and a methyl group that packs against the hydrophobic pocket ofubiquitin formed by Leu-8, Ile-44, His-68, and Val-70.

• Piserchio A et al.
• The PDZ1 domain of SAP90. Characterization of structure and binding.
• J Biol Chem. 2002; 277: 6967-73
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• The structural features of the PDZ1 domain of the synapse-associatedprotein SAP90 have been characterized by NMR. A comparison with thestructures of the PDZ2 and PDZ3 domains of SAP90 illustrates significantdifferences, which may account for the unique binding properties of thesehomologous domains. Within the postsynaptic density, SAP90 functions as amolecular scaffold with a number of the protein-protein interactionsmediated through the PDZ1 domain. Here, using fluorescence anisotropy andNMR chemical shift analysis, we have characterized the association of PDZ1to the C-terminal peptides of the GluR6 subunit of the kainate receptor,voltage-gated K(+) channel Kv1.4, and microtubule-associate protein CRIPT,all of which are known to associate with SAP90. The latter two, whichpossess the consensus sequence for binding to PDZ domains (T/S-X-V-oh),have low micromolar binding affinities (1.5-15 microm). The C terminus ofGluR6, RLPGKETMA-oh, lacking the consensus sequence, binds to PDZ1 ofSAP90 with an affinity of 160 microm. The NMR data illustrate thatalthough all three peptides occupy the binding groove capped by the GLGFloop of PDZ1, specific differences are present, consistent with thevariation in binding affinities.

• Misra S, Beach BM, Hurley JH
• Structure of the VHS domain of human Tom1 (target of myb 1): insights intointeractions with proteins and membranes.
• Biochemistry. 2000; 39: 11282-90
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• VHS domains are found at the N-termini of select proteins involved inintracellular membrane trafficking. We have determined the crystalstructure of the VHS domain of the human Tom1 (target of myb 1) protein to1.5 A resolution. The domain consists of eight helices arranged in asuperhelix. The surface of the domain has two main features: (1) a basicpatch on one side due to several conserved positively charged residues onhelix 3 and (2) a negatively charged ridge on the opposite side, formed byresidues on helix 2. We compare our structure to the recently obtainedstructure of tandem VHS-FYVE domains from Hrs [Mao, Y., Nickitenko, A.,Duan, X., Lloyd, T. E., Wu, M. N., Bellen, H., and Quiocho, F. A. (2000)Cell 100, 447-456]. Key features of the interaction surface between theFYVE and VHS domains of Hrs, involving helices 2 and 4 of the VHS domain,are conserved in the VHS domain of Tom1, even though Tom1 does not have aFYVE domain. We also compare the structures of the VHS domains of Tom1 andHrs to the recently obtained structure of the ENTH domain of epsin-1[Hyman, J., Chen, H., Di Fiore, P. P., De Camilli, P., and Brunger, A. T.(2000) J. Cell Biol. 149, 537-546]. Comparison of the two VHS domains andthe ENTH domain reveals a conserved surface, composed of helices 2 and 4,that is utilized for protein-protein interactions. In addition, VHSdomain-containing proteins are often localized to membranes. We suggestthat the conserved positively charged surface of helix 3 in VHS and ENTHdomains plays a role in membrane binding.

• Ohta T, Michel JJ, Schottelius AJ, Xiong Y
• ROC1, a homolog of APC11, represents a family of cullin partners with anassociated ubiquitin ligase activity.
• Mol Cell. 1999; 3: 535-41
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• We have identified two highly conserved RING finger proteins, ROC1 andROC2, that are homologous to APC11, a subunit of the anaphase-promotingcomplex. ROC1 and ROC2 commonly interact with all cullins while APC11specifically interacts with APC2, a cullin-related APC subunit. YeastROC1encodes an essential gene whose reduced expression resulted in multiple,elongated buds and accumulation of Sic1p and Cln2p. ROC1 and APC11immunocomplexes can catalyze isopeptide ligations to form polyubiquitinchains in an E1- and E2-dependent manner. ROC1 mutations completelyabolished their ligase activity without noticeable changes in associatedproteins. Ubiquitination of phosphorylated I kappa B alpha can becatalyzed by the ROC1 immunocomplex in vitro. Hence, combinations ofROC/APC11 and cullin proteins proteins potentially constitute a widevariety of ubiquitin ligases.

• Yaron A et al.
• Inhibition of NF-kappa-B cellular function via specific targeting of theI-kappa-B-ubiquitin ligase.
• EMBO J. 1997; 16: 6486-94
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• Activation of the transcription factor NF-kappa B is a paradigm for signaltransduction through the ubiquitin-proteasome pathway: ubiquitin-dependentdegradation of the transcriptional inhibitor I kappa B in response to cellstimulation. A major issue in this context is the nature of therecognition signal and the targeting enzyme involved in the proteolyticprocess. Here we show that following a stimulus-dependent phosphorylation,and while associated with NF-kappa B, I kappa B is targeted by a specificubiquitin-ligase via direct recognition of the signal-dependentphosphorylation site; phosphopeptides corresponding to this sitespecifically inhibit ubiquitin conjugation of I kappa B and its subsequentdegradation. The ligase recognition signal is functionally conservedbetween I kappa B alpha and I kappa B beta, and does not involve thenearby ubiquitination site. Microinjection of the inhibitory peptides intostimulated cells abolished NF-kappa B activation in response to TNF alphaand the consequent expression of E-selectin, an NF-kappa B-dependentcell-adhesion molecule. Inhibition of NF-kappa B function by specificblocking of ubiquitin ligase activity provides a novel approach forintervening in cellular processes via regulation of unique proteolyticevents.

• Varshavsky A
• The N-end rule.
• Cell. 1992; 69: 725-35