Volume 37, Issue 1, Pages 102-111 (January 2010) Substrate-Assisted Inhibition of Ubiquitin-like Protein-Activating Enzymes: The NEDD8 E1 Inhibitor MLN4924 Forms a NEDD8-AMP Mimetic In Situ  James E. Brownell, Michael D. Sintchak, James M. Gavin, Hua Liao, Frank J. Bruzzese, Nancy J. Bump, Teresa A. Soucy, Michael A. Milhollen, Xiaofeng Yang, Anne L. Burkhardt, Jingya Ma, Huay-Keng Loke, Trupti Lingaraj, Dongyun Wu, Kristin B. Hamman, James J. Spelman, Courtney A. Cullis, Steven P. Langston, Stepan Vyskocil, Todd B. Sells, William D. Mallender, Irache Visiers, Ping Li, Christopher F. Claiborne, Mark Rolfe, Joseph B. Bolen, Lawrence R. Dick  Molecular Cell  Volume 37, Issue 1, Pages 102-111 (January 2010) DOI: 10.1016/j.molcel.2009.12.024 Copyright © 2010 Elsevier Inc. Terms and Conditions

Figure 1 Structure of the NEDD8-MLN4924 Adduct Bound to NAE (A) The chemical structure of MLN4924. (B) A 3.0 Å sigmaA-weighted 2Fo − Fc electron density map contoured at 1.0σ covering the NEDD8-MLN4924 adduct occupying the adenylation domain of NAE. Carbon is shown in yellow (MLN4924) or green (NEDD8); nitrogen, blue; oxygen, red; and sulfur, orange. NAEβ is shown with helices in cyan, β-strands in magenta, and loops in salmon. (C) High-resolution mass spectrum and fragment ion spectrum of Gly-Gly-MLN4924 (right) and assignment of product ions (left). (D) NAE active site view with NEDD8-ATP (PDB entry 1R4N, left) or NEDD8-MLN4924 adduct (right) bound. NAE amino acids are numbered according to UniProtKB/Swiss-Prot entry Q8TBC4 (UBA3_HUMAN). Hydrogen bonds are indicated by dashed lines. Atoms are colored as in (A) except that carbon is salmon (ATP), and phosphate is orange. See also Figure S1. Molecular Cell 2010 37, 102-111DOI: (10.1016/j.molcel.2009.12.024) Copyright © 2010 Elsevier Inc. Terms and Conditions

Figure 2 Mechanism of NAE-Catalyzed Adduct Formation (A) Mass spectra of reactions containing NEDD8 and MgATP with wild-type NAE (top) or NAEβ(C237A) (bottom) in the presence (red traces) or absence (blue traces) of MLN4924. (B) NAE and NEDD8 were incubated without (lanes 1 and 2) or with 10 μM MLN4924 (lanes 3–6). Inhibitor was added before (“b,” lanes 3 and 4) or after (“a,” lanes 5 and 6) MgATP, followed by addition of 20 mM EDTA. UBC12 was added in lanes 2, 4, and 6. Samples were processed for western blotting under nonreducing conditions and probed with anti-NEDD8 (top) or anti-MLN4924 antibodies (bottom); NEDD8-AMP was detected by autoradiography (middle). (C) Schematic showing the mechanism of NEDD8-MLN4924 adduct formation. See also Figure S3. Molecular Cell 2010 37, 102-111DOI: (10.1016/j.molcel.2009.12.024) Copyright © 2010 Elsevier Inc. Terms and Conditions

Figure 3 NEDD8-MLN4924 Is a Tight-Binding Inhibitor of NAE (A) Dose response of MLN4924 inhibition of ATP-PPi exchange reactions using wild type NAE (triangles) or NAEβ(C237A) (circles). (B) Scheme describing mechanism-based inhibition of NAE by MLN4924. (C) The rate constant for NAE inactivation as a function of ATP concentration. The data were fit to a binding isotherm: kobs/[I] = (kinact/Ki)/(1 + [ATP]/KM). (D) Progress curves for UBC12 transthiolation reactions using NAE (circles), NAE:APN (squares), and NAE:NEDD8-MLN4924 (triangles). Reactions contained 50 μM (open symbols) or 3 mM (solid symbols) ATP. The error bars in (A) and (D) represent the standard deviation of three independent experiments. See also Figure S4. Molecular Cell 2010 37, 102-111DOI: (10.1016/j.molcel.2009.12.024) Copyright © 2010 Elsevier Inc. Terms and Conditions

Figure 4 NEDD8-MLN4924 Adduct Formation in Treated Cells (A) HCT116 cells were treated with 1 μM MLN4924 and harvested at the time points indicated. Cell lysates were processed for western blotting under nonreducing conditions and probed with anti-NEDD8 (top) and anti-MLN4924 antibodies (bottom). The asterisk indicates a nonspecific band. (B) HCT116 cells were treated with vehicle (lanes 1, 2, 5, and 6) or 3 μM MLN4924 (lanes 3, 4, 7, and 8) for 2 hr and then placed in fresh medium without inhibitor. Samples were removed at time 0 (lanes 1–4) or 4 hours (lanes 5–8) postwashout. Cells were also treated without (odd numbered lanes) or with 70 μM cycloheximide (CHX; even numbered lanes) throughout the experiment. Lysates were processed as in (A). Molecular Cell 2010 37, 102-111DOI: (10.1016/j.molcel.2009.12.024) Copyright © 2010 Elsevier Inc. Terms and Conditions

Figure 5 E1 Catalyzed UBL-Inhibitor Adduct Formation Mass spectrometry of UBL-adduct-forming reactions with compound 1 incubated in the presence of EDTA (blue traces) or MgATP (red traces). (A–F) The mass of each UBL and UBL-compound 1 adduct is indicated. Note that, in (F), a significant amount of GABARAP-AMP was detected in addition to GABARAP-compound 1. (A) UAE/Ub. (B) NAE/NEDD8. (C) UBA6/Ub. (D) SAE/SUMO1. (E) UBA7/ISG15. (F) ATG7/GABARAP. (G) The chemical structure of compound 1. (H) The structure of the UBL-compound 1 adduct. Molecular Cell 2010 37, 102-111DOI: (10.1016/j.molcel.2009.12.024) Copyright © 2010 Elsevier Inc. Terms and Conditions