Volume 21, Issue 1, Pages 42-53 (January 2013) Structural Conservation of Distinctive N-terminal Acetylation-Dependent Interactions across a Family of Mammalian NEDD8 Ligation Enzymes  Julie K. Monda, Daniel C. Scott, Darcie J. Miller, John Lydeard, David King, J. Wade Harper, Eric J. Bennett, Brenda A. Schulman  Structure  Volume 21, Issue 1, Pages 42-53 (January 2013) DOI: 10.1016/j.str.2012.10.013 Copyright © 2013 Elsevier Ltd Terms and Conditions

Figure 1 Role of N-terminal Acetylation of the NEDD8 E2 UBE2F in Human DCNL PONY Domain-Stimulated NEDD8 Ligation (A) MS/MS spectrum resulting from the N-terminal peptide after Arg-C digestion/desalting of human UBE2F expressed in NIH 3T3 cells following affinity purification via a C-terminal tag. The corresponding XCorr and ΔCN values are indicated as well as the y (red) and b (blue) ions used to match the peptide sequence. (B) MS/MS spectrum of the N-terminal peptide from Arg-C digested UBE2F-His purified from insect cells upon baculovirus-mediated expression. (C) Pulse-chase [32P]∼NEDD8 transfer from unacetylated UBE2F to either CUL1CTD-RBX1 (labeled CUL1C-RBX1) or CUL5CTD-RBX2 (labeled CUL5C-RBX2) in the absence or presence of the indicated DCNL PONY domain. (D) Pulse-chase [32P]∼NEDD8 transfer assays as in (C), except with the N-terminally acetylated E2 UBE2FNAc. See also Figure S1. Structure 2013 21, 42-53DOI: (10.1016/j.str.2012.10.013) Copyright © 2013 Elsevier Ltd Terms and Conditions

Figure 2 Human DCNL PONY Domain Interactions with Different Cullins and N-terminally Acetylated NEDD8 E2s (A) Thermodynamic parameters determined by ITC for binding between the indicated human DCNL PONY domains and cullin WHB subdomains. (B) Previously published structure of CUL1WHB (green) - DCNL1P(salmon) (PDB ID code 3TDU; UBC12NAc peptide not shown) complex (Scott et al., 2011) and model of CUL5WHB (olive) (Duda et al., 2008)- DCNL1P(salmon) (Scott et al., 2011), highlighting DCNL-CUL interacting residues. (C) Thermodynamic parameters determined by ITC for binding between the indicated human DCNL PONY domains and unacetylated and N-terminally acetylated versions of UBE2F, and an N-terminally acetylated peptide corresponding to UBC12. See also Figure S2. Structure 2013 21, 42-53DOI: (10.1016/j.str.2012.10.013) Copyright © 2013 Elsevier Ltd Terms and Conditions

Figure 3 Variations in DCNL PONY Domain Activation of NEDD8 Transfer from N-terminally Acetylated NEDD8 E2s to Different Cullin C-terminal Domain/RBX Complexes (A) As indicated in the schematic diagram, pulse-chase [32P]∼NEDD8 transfer from UBC12NAc (left) or UBE2FNAc (right) to the indicated cullin C-terminal domain-RBX complexes in the absence or presence of the indicated DCNL PONY domain. For comparison, all reactions were carried out under the same conditions. (B) Extended time courses from (A) for pulse-chase [32P]∼NEDD8 transfer from UBE2FNAc to either CUL3CTD-RBX1 or CUL4ACTD-RBX1 in the absence or presence of the indicated DCNL PONY domain. (C) Pulse-chase reactions with the indicated concentrations of various components, monitoring [32P]∼NEDD8 transfer from UBE2FNAc to CUL5CTD-RBX2 in the absence or presence of the PONY domain from DCNL1 or DCNL3. Structure 2013 21, 42-53DOI: (10.1016/j.str.2012.10.013) Copyright © 2013 Elsevier Ltd Terms and Conditions

Figure 4 Peptide Inhibition of DCNL Activation of NEDD8 Ligation Depends on N-terminal Acetylation (A) ITC data for interactions between DCNL3P and unacetylated or N-terminally acetylated peptides from UBE2F. Upper panels show raw power data recorded during titration experiments, and lower panels show fits of standard binding equations after integration of the raw data, using Origin (v. 7.0) software provided from MicroCal. (B) Pulse-chase monitoring [32P]∼NEDD8 transfer from UBE2FNAc to CUL5CTD-RBX2 in the absence or presence of the PONY domain from DCNL1 or DCNL3, and unacetylated or N-terminally acetylated peptides corresponding to UBC12 or UBE2F. Structure 2013 21, 42-53DOI: (10.1016/j.str.2012.10.013) Copyright © 2013 Elsevier Ltd Terms and Conditions

Figure 5 Overall Conserved Mode of DCNL PONY Domain Interactions with Acetylated N-terminal Helices from NEDD8 E2s (A) Cartoon representation of overall structure of DCNL2 PONY domain (raspberry) complex with peptide from UBC12NAc (teal) with N-acetyl-Met1, Ile2, and Leu4 shown in sticks. (B) Cartoon representation of overall structure of DCNL3 PONY domain (brick) complex with peptide from UBE2FNAc (lime) with N-acetyl-Met1, Leu2, and Leu4 shown in sticks. (C) Superposition of DCNL2P-UBC12NAc1-12 and DCNL3P-UBE2FNAc1-25 crystal structures with prior structure of DCNL1P (pink)-UBC12NAc1-15 (cyan) (PDB ID code 3TDU) (Scott et al., 2011). See also Figure S3. Structure 2013 21, 42-53DOI: (10.1016/j.str.2012.10.013) Copyright © 2013 Elsevier Ltd Terms and Conditions

Figure 6 A DCNL Hydrophobic Pocket Surrounds N-acetyl-methionine from a NEDD8 E2 (A) Close-up view of DCNL2P-UBC12NAc1-12 structure, with DCNL2P surface colored by electrostatic potential, and UBC12NAc1-12 shown in cyan with N-acetyl-Met1, Ile2, and Leu4 shown in sticks. (B) Close-up view of DCNL3P-UBE2FNAc1-25 structure, with DCNL3P surface colored by electrostatic potential, and UBE2FNAc1-25 shown in cyan with N-acetyl-Met1, Leu2, and Leu4 shown in sticks. See also Figure S4. Structure 2013 21, 42-53DOI: (10.1016/j.str.2012.10.013) Copyright © 2013 Elsevier Ltd Terms and Conditions

Figure 7 Amino Acid Identity Downstream of N-acetyl-methionine Influences E2 Specificity of DCNL-Stimulated NEDD8 Ligation (A) Close-up views of superimposed DCNL1P-UBC12NAc1-15 (Scott et al., 2011) and DCNL3P-UBE2FNAc1-25 crystal structures, highlighting interactions with residues downstream of the N terminus. (B) Pulse-chase assays monitoring [32P]∼NEDD8 transfer from UBC12NAc (top) or UBE2FNAc (lower) to CUL2CTD-RBX1 in the absence or presence of the wild-type and indicated mutant versions of the PONY domains from DCNL1 and DCNL3. Structure 2013 21, 42-53DOI: (10.1016/j.str.2012.10.013) Copyright © 2013 Elsevier Ltd Terms and Conditions