Dcn1 Functions as a Scaffold-Type E3 Ligase for Cullin Neddylation Thimo Kurz, Yang-Chieh Chou, Andrew R. Willems, Nathalie Meyer-Schaller, Marie-Lyn Hecht, Mike Tyers, Matthias Peter, Frank Sicheri  Molecular Cell  Volume 29, Issue 1, Pages 23-35 (January 2008) DOI: 10.1016/j.molcel.2007.12.012 Copyright © 2008 Elsevier Inc. Terms and Conditions

Figure 1 Structure of ScDcn1 (A) Ribbon representation of full-length S. cerevisiae Dcn1. The UBA and PONY domains are shown in red and blue, respectively. (B) ScDcn1 forms head-to-tail dimers in the crystal environment. (C) Surface representation of ScDcn1 highlighting the dimer contact surfaces. UBA and PONY domains are colored pink and gray, respectively, with UBA to PONY domain contact surfaces shown in red and PONY domain to PONY domain contact surfaces shown in blue. (D) Overlay of the ScDcn1 UBA domain in red, with the human Tap/NXF1 UBA domain in green. (E) Apparent solution molecular weights of the indicated Dcn1 proteins derived by analytical ultracentrifugation. (F) Full-length HA-tagged Dcn1 and HA-tagged Dcn1Δ69 lacking the UBA domain efficiently promote neddylation of endogenous Cdc53 when expressed in dcn1Δ yeast cells. Molecular Cell 2008 29, 23-35DOI: (10.1016/j.molcel.2007.12.012) Copyright © 2008 Elsevier Inc. Terms and Conditions

Figure 2 Functional Analysis of the EF-Hand-like Elements in the PONY Domain of Dcn1 (A) Structural overlay of the paired EF-Hand-like elements of Dcn1 (blue) with the EF-Hand elements of human Cbl (orange) and human Mts1 (pink). (B) Sequence comparison of Dcn1 EF-Hand-like elements 2 and 3 (EF-Hand 2 and EF-Hand 3) with the EF-Hand elements of Cbl and Mts1. Positions that participate in Ca2+ binding are shown in red. Possible calcium-binding residues in Dcn1 are highlighted with a red box. (C) In vivo kinetics of cullin neddylation upon induction of Dcn1 expression. Wild-type Dcn1 or the indicated EF-Hand mutants (EF-Hand 2: K119G E122G E123G K126G; EF-Hand 3: D176G N178G K180G) were expressed from a galactose-inducible promoter in dcn1Δ yeast. The kinetics of Cdc53 neddylation were assayed following galactose induction in 10 min intervals by immunoblot. Molecular Cell 2008 29, 23-35DOI: (10.1016/j.molcel.2007.12.012) Copyright © 2008 Elsevier Inc. Terms and Conditions

Figure 3 Dcn1 Directly Binds to Cullins through the DAD Patch (A) The DAD patch is conserved within the C-terminal region of the Dcn1 PONY domain. Zoom-in view displays the net acidic and hydrophobic nature of the DAD patch. (B) Immunoblot analysis of the effect of the indicated DAD patch mutations in Dcn1 on Cdc53 neddylation in vivo. (C) Quantitative analysis of two-hybrid interaction between Cdc53 and wild-type Dcn1 or the indicated Dcn1 mutants. Error bars represent standard deviation of β-galactosidase activty for at least three independent experiments. (D) GST pull-down analysis of the interaction between an Sf9-expressed Myc-Cdc53/GST-Rbx1 complex and bacterially expressed wild-type His6-Dcn1 or the indicated Dcn1 mutants. (E) GST pull-down analysis of the interaction between a bacterially expressed His6-Cul1324–776/GST-Roc1 complex and wild-type His6-hDCNL1 or the indicated hDCNL1 mutants. Molecular Cell 2008 29, 23-35DOI: (10.1016/j.molcel.2007.12.012) Copyright © 2008 Elsevier Inc. Terms and Conditions

Figure 4 Dcn1 Binds Cullins on a Highly Conserved Surface in Close Proximity to the Neddylated Lysine (A) Sequence alignment of yeast Cdc53 with human cullins. Conserved charged residues, which were mutated to alanines, are shaded. Brackets indicate the mutant combinations analyzed, which were named according to the mutated residues, followed by the position of the first residue in the cluster. Asterisk denotes the known/projected site of neddylation. (B) Anti-Myc immunoblot analysis of whole-cell extracts from yeast strains expressing wild-type untagged Cdc53 or Myc-tagged Cdc53 or a K760R Cdc53 mutant. (C) Synthetic growth defects of a Cdc53-K760R mutant with the indicated mutant components of the SCF ubiquitination system. (D) Coimmunoprecipitation analysis of Cdc34, Skp1, and Rbx1 with wild-type Cdc53 or a Cdc53-K760R mutant. (E) Two-hybrid analysis of binding between Dcn1 and wild-type Cdc53 and the indicated Cdc53 mutants. Error bars represent standard deviation of β-galactosidase activty for at least three independent experiments. (F) Effect of R804 and KRD790 mutations on Cdc53 neddylation in vivo (a) and on Cdc53 binding to bacterially expressed Dcn1 in vitro using a GST pull-down assay (b). (G) The Dcn1-binding surface on Cdc53 is conserved across all cullins. A homology model of Cdc53 (shown as surface representation) bound to Rbx1 (shown in green ribbon) was generated from the human Cul1/Roc1 complex structure (PDB ID, 1LDK) using Swiss PDB model. KRD790 and R804 Cdc53 mutations map to a conserved surface centered 18 Å from the K760 site of neddylation (inset). (H) Mutational analysis of binding between a C-terminal fragment of human Cul1, hDcnl1 (the human ortholog of ScDcn1), and GST-tagged Roc1 (the human ortholog of ScRbx1). All proteins were expressed and purified from bacteria. Molecular Cell 2008 29, 23-35DOI: (10.1016/j.molcel.2007.12.012) Copyright © 2008 Elsevier Inc. Terms and Conditions

Figure 5 Dcn1 Binds Directly and Specifically to Ubc12 (A) GST pull-down analysis of interaction between recombinant bacterially expressed GST-Dcn1 and endogenously expressed HA-tagged Ubc12. Binding of GST-Dcn1 to Ubc12-HA was detected by immunoblot with an anti-HA antibody (band highlighted by asterisk). (B) GST pull-down analysis of binding between the indicated GST-tagged E2 enzymes and His6-ScDcn1. All proteins were expressed and purified from bacteria. Loading controls for His-Dcn1 and GST-fused protein levels are in the bottom two panels. (C) GST pull-down analysis of binding between His6-hDCNL1 and wild-type and the indicated mutant forms of GST-tagged hUbc12. GST-UbcH7 serves as negative control. All proteins were expressed and purified from bacteria. Molecular Cell 2008 29, 23-35DOI: (10.1016/j.molcel.2007.12.012) Copyright © 2008 Elsevier Inc. Terms and Conditions

Figure 6 In Vitro Reconstitution of the Cullin Neddylation Reaction (A) In vitro neddylation of an Sf9-expressed and -purified Myc-Cdc53/His6-Rbx1 complex. In vitro reactions were performed with dcn1Δ yeast extracts and the indicated levels of E2 enzyme. Cullin neddylation was assessed by immunoblot using a Myc antibody. (B) In vitro neddylation of an Sf9-produced Myc-Cdc53/His6-Rbx1 complex in the presence of low E2 concentrations and the indicated quantities of bacterially expressed GST-Dcn1 and/or whole-cell extracts prepared from wild-type or dcn1Δ yeast strains. (C) Analysis of the requirement of the UBA domain of Dcn1 for in vitro neddylation of Cdc53 in the presence of low E2 amounts, using either recombinant full-length Dcn1 or Dcn1Δ69. (D) Functional analysis of the DAD patch of Dcn1. Reactions were performed as in (B), using 30 ng of purified bacterially expressed wild-type Dcn1 or a D226A/A253R/D259A mutant. (E) Kinetic analysis of cullin neddylation as function of increasing amounts of Dcn1. Reactions were carried out as in (B), with changes to the concentration of Dcn1 as indicated. (F) Extract-independent reconstitution of Cdc53 neddylation. Reactions were carried out as in (C), with the indicated variation in the amount of recombinant Dcn1 used. (G) Ribbon representation of ScDcn1 highlighting the position of cysteine residues mutated in this study. (H) Functional analysis of cysteine mutations in Dcn1 on Cdc53 neddylation in vivo. The indicated mutant versions of HA-Dcn1 were reintroduced into dcn1Δ yeast strain, and neddylation of endogenous Cdc53 was monitored by immunoblotting (top panel). Immunoblotting with HA antibodies (lower panel) confirms equal expression of the mutated proteins. Molecular Cell 2008 29, 23-35DOI: (10.1016/j.molcel.2007.12.012) Copyright © 2008 Elsevier Inc. Terms and Conditions