Rapid E2-E3 Assembly and Disassembly Enable Processive Ubiquitylation of Cullin-RING Ubiquitin Ligase Substrates

Degradation by the ubiquitin-proteasome system requires assembly of a polyubiquitin chain upon substrate. However, the structural and mechanistic features that enable template-independent processive chain synthesis are unknown. We show that chain assembly by ubiquitin ligase SCF and ubiquitin-conjugating enzyme Cdc34 is facilitated by the unusual nature of Cdc34-SCF transactions: Cdc34 binds SCF with nanomolar affinity, nevertheless the complex is extremely dynamic. These properties are enabled by rapid association driven by electrostatic interactions between the acidic tail of Cdc34 and a basic 'canyon' in the Cul1 subunit of SCF. Ab initio docking between Cdc34 and Cul1 predicts intimate contact between the tail and the basic canyon, an arrangement confirmed by crosslinking and kinetic analysis of mutants. Basic canyon residues are conserved in both Cul1 paralogs and orthologs, suggesting that the same mechanism underlies processivity for all cullin-RING ubiquitin ligases. We discuss different strategies by which processive ubiquitin chain synthesis may be achieved.

[1]  R. Deshaies,et al.  UBXD7 Binds Multiple Ubiquitin Ligases and Implicates p97 in HIF1α Turnover , 2008, Cell.

[2]  Martin Rechsteiner,et al.  Recognition of the polyubiquitin proteolytic signal , 2000, The EMBO journal.

[3]  R. Osman,et al.  Autoinhibitory regulation of SCF-mediated ubiquitination by human cullin 1's C-terminal tail , 2008, Proceedings of the National Academy of Sciences.

[4]  Ping Wang,et al.  Structure of a c-Cbl–UbcH7 Complex RING Domain Function in Ubiquitin-Protein Ligases , 2000, Cell.

[5]  A. Fersht.  Structure and mechanism in protein science , 1998 .

[6]  Jeffrey J. Gray,et al.  Protein-protein docking with simultaneous optimization of rigid-body displacement and side-chain conformations. , 2003, Journal of molecular biology.

[7]  M. Tyers,et al.  Suprafacial Orientation of the SCFCdc4 Dimer Accommodates Multiple Geometries for Substrate Ubiquitination , 2007, Cell.

[8]  Huan‐Xiang Zhou,et al.  Electrostatic rate enhancement and transient complex of protein–protein association , 2008, Proteins.

[9]  Anjanabha Saha,et al.  Multimodal activation of the ubiquitin ligase SCF by Nedd8 conjugation , 2008, Molecular cell.

[10]  M. Goebl,et al.  A chimeric ubiquitin conjugating enzyme that combines the cell cycle properties of CDC34 (UBC3) and the DNA repair properties of RAD6 (UBC2): implications for the structure, function and evolution of the E2s. , 1992, The EMBO journal.

[11]  Nurhan Özlü,et al.  The conserved protein DCN-1/Dcn1p is required for cullin neddylation in C. elegans and S. cerevisiae , 2005, Nature.

[12]  A. Varshavsky,et al.  Ubiquitin as a degradation signal. , 1992, The EMBO journal.

[13]  R Hodgins,et al.  Functional and physical characterization of the cell cycle ubiquitin-conjugating enzyme CDC34 (UBC3). Identification of a functional determinant within the tail that facilitates CDC34 self-association. , 1994, The Journal of biological chemistry.

[14]  Brian Kuhlman,et al.  E2 conjugating enzymes must disengage from their E1 enzymes before E3-dependent ubiquitin and ubiquitin-like transfer , 2005, Nature Structural &Molecular Biology.

[15]  S. Jentsch,et al.  Regulation of apoptosis and cytokinesis by the anti-apoptotic E2/E3 ubiquitin-ligase BRUCE. , 2008, Ernst Schering Foundation symposium proceedings.

[16]  R. Deshaies,et al.  In vitro reconstitution of SCF substrate ubiquitination with purified proteins. , 2005, Methods in enzymology.

[17]  T. Creighton.  Proteins: Structures and Molecular Properties , 1986 .

[18]  A. Fersht,et al.  Rapid, electrostatically assisted association of proteins , 1996, Nature Structural Biology.

[19]  Gideon Schreiber,et al.  Fundamental Aspects of Protein—Protein Association Kinetics , 2009 .

[20]  R. Deshaies,et al.  RING domain E3 ubiquitin ligases. , 2009, Annual review of biochemistry.

[21]  D. C. Dias,et al.  Nedd8 on cullin: building an expressway to protein destruction , 2004, Oncogene.

[22]  S. Elledge,et al.  Structure of the Cul1–Rbx1–Skp1–F boxSkp2 SCF ubiquitin ligase complex , 2002, Nature.

[23]  D. Ecker,et al.  A multiubiquitin chain is confined to specific lysine in a targeted short-lived protein. , 1989, Science.

[24]  M. Tyers,et al.  Structural Basis for Phosphodependent Substrate Selection and Orientation by the SCFCdc4 Ubiquitin Ligase , 2003, Cell.

[25]  Robert C. Edgar,et al.  MUSCLE: multiple sequence alignment with high accuracy and high throughput. , 2004, Nucleic acids research.

[26]  Nathan A. Baker,et al.  Electrostatics of nanosystems: Application to microtubules and the ribosome , 2001, Proceedings of the National Academy of Sciences of the United States of America.

[27]  D. Wolf,et al.  Degradation of misfolded protein in the cytoplasm is mediated by the ubiquitin ligase Ubr1 , 2008, FEBS letters.

[28]  J Wade Harper,et al.  Structure of a Fbw7-Skp1-cyclin E complex: multisite-phosphorylated substrate recognition by SCF ubiquitin ligases. , 2007, Molecular cell.

[29]  Amanda Doucette,et al.  An inhibitor of NEDD8-activating enzyme as a new approach to treat cancer , 2009, Nature.

[30]  A. Shevchenko,et al.  Promotion of NEDD8-CUL1 Conjugate Cleavage by COP9 Signalosome , 2001, Science.

[31]  N. Pavletich,et al.  High-level expression and purification of recombinant SCF ubiquitin ligases. , 2005, Methods in enzymology.

[32]  H. Gutfreund,et al.  Proteins: structures and molecular properties (second edition): T.E. Creighton, Freeman; New York, 1992; x + 507 pages; £22.95. ISBN 0-7167-7030-X , 1993 .

[33]  M. Roussel,et al.  E2-RING expansion of the NEDD8 cascade confers specificity to cullin modification. , 2009, Molecular cell.

[34]  J. Holton,et al.  Structural basis for recruitment of Ubc12 by an E2 binding domain in NEDD8's E1. , 2005, Molecular cell.

[35]  D. Baker,et al.  Design of a Novel Globular Protein Fold with Atomic-Level Accuracy , 2003, Science.

[36]  David Baker,et al.  Macromolecular modeling with rosetta. , 2008, Annual review of biochemistry.

[37]  G Schreiber,et al.  Interaction of barnase with its polypeptide inhibitor barstar studied by protein engineering. , 1993, Biochemistry.

[38]  Christian Cole,et al.  The Jpred 3 secondary structure prediction server , 2008, Nucleic Acids Res..

[39]  V. Kirkin,et al.  Role of ubiquitin- and Ubl-binding proteins in cell signaling. , 2007, Current opinion in cell biology.

[40]  Tony Pawson,et al.  Multisite phosphorylation of a CDK inhibitor sets a threshold for the onset of DNA replication , 2001, Nature.

[41]  Gideon Schreiber,et al.  Fruitful and futile encounters along the association reaction between proteins. , 2009, Biophysical journal.

[42]  Raymond J. Deshaies,et al.  Mechanism of Lysine 48-Linked Ubiquitin-Chain Synthesis by the Cullin-RING Ubiquitin-Ligase Complex SCF-Cdc34 , 2005, Cell.

[43]  A. Varshavsky,et al.  The E2–E3 interaction in the N‐end rule pathway: the RING‐H2 finger of E3 is required for the synthesis of multiubiquitin chain , 1999, The EMBO journal.

[44]  Wei Xu,et al.  Editing of ubiquitin conjugates by an isopeptidase in the 26S proteasome , 1997, Nature.

[45]  M. Goebl,et al.  An Essential Domain within Cdc34p Is Required for Binding to a Complex Containing Cdc4p and Cdc53p in Saccharomyces cerevisiae * , 1998, The Journal of Biological Chemistry.

[46]  David O. Morgan,et al.  Sequential E2s Drive Polyubiquitin Chain Assembly on APC Targets , 2007, Cell.

[47]  Kim Nasmyth,et al.  The B-type cyclin kinase inhibitor p40 SIC1 controls the G1 to S transition in S. cerevisiae , 1994, Cell.

[48]  S. Gygi,et al.  Ubiquitin Chains Are Remodeled at the Proteasome by Opposing Ubiquitin Ligase and Deubiquitinating Activities , 2006, Cell.

[49]  J. Tóth,et al.  Identification of a portable determinant of cell cycle function within the carboxyl‐terminal domain of the yeast CDC34 (UBC3) ubiquitin conjugating (E2) enzyme. , 1992, The EMBO journal.

[50]  Daniel C. Scott,et al.  Structural Insights into NEDD8 Activation of Cullin-RING Ligases: Conformational Control of Conjugation , 2008, Cell.

[51]  Wei Li,et al.  A ubiquitin ligase transfers preformed polyubiquitin chains from a conjugating enzyme to a substrate , 2007, Nature.

[52]  R. Norel,et al.  Electrostatic aspects of protein-protein interactions. , 2000, Current opinion in structural biology.

[53]  A. Weissman,et al.  The activity of a human endoplasmic reticulum-associated degradation E3, gp78, requires its Cue domain, RING finger, and an E2-binding site. , 2006, Proceedings of the National Academy of Sciences of the United States of America.

[54]  S. Reed,et al.  Human F-box protein hCdc4 targets cyclin E for proteolysis and is mutated in a breast cancer cell line , 2001, Nature.

[55]  David Baker,et al.  Protein Structure Prediction Using Rosetta , 2004, Numerical Computer Methods, Part D.