Insights into E3 ligase activity revealed by a SUMO–RanGAP1–Ubc9–Nup358 complex

SUMO-1 (for small ubiquitin-related modifier) belongs to the ubiquitin (Ub) and ubiquitin-like (Ubl) protein family. SUMO conjugation occurs on specific lysine residues within protein targets, regulating pathways involved in differentiation, apoptosis, the cell cycle and responses to stress by altering protein function through changes in activity or cellular localization or by protecting substrates from ubiquitination. Ub/Ubl conjugation occurs in sequential steps and requires the concerted action of E2 conjugating proteins and E3 ligases. In addition to being a SUMO E3, the nucleoporin Nup358/RanBP2 localizes SUMO-conjugated RanGAP1 to the cytoplasmic face of the nuclear pore complex by means of interactions in a complex that also includes Ubc9, the SUMO E2 conjugating protein. Here we describe the 3.0-Å crystal structure of a four-protein complex of Ubc9, a Nup358/RanBP2 E3 ligase domain (IR1-M) and SUMO-1 conjugated to the carboxy-terminal domain of RanGAP1. Structural insights, combined with biochemical and kinetic data obtained with additional substrates, support a model in which Nup358/RanBP2 acts as an E3 by binding both SUMO and Ubc9 to position the SUMO–E2-thioester in an optimal orientation to enhance conjugation.

[1]  M. Dasso,et al.  RanBP2 associates with Ubc9p and a modified form of RanGAP1. , 1997, Proceedings of the National Academy of Sciences of the United States of America.

[2]  P. Hieter,et al.  The APC11 RING-H2 finger mediates E2-dependent ubiquitination. , 2000, Molecular biology of the cell.

[3]  T. Sixma,et al.  The RanBP2 SUMO E3 ligase is neither HECT- nor RING-type , 2004, Nature Structural &Molecular Biology.

[4]  G. Blobel,et al.  A novel ubiquitin-like modification modulates the partitioning of the Ran-GTPase-activating protein RanGAP1 between the cytosol and the nuclear pore complex , 1996, The Journal of cell biology.

[5]  A. Dejean,et al.  The Nucleoporin RanBP2 Has SUMO1 E3 Ligase Activity , 2002, Cell.

[6]  Richard S. Rogers,et al.  A conserved catalytic residue in the ubiquitin‐conjugating enzyme family , 2003, The EMBO journal.

[7]  C. Lima,et al.  Structures of the SUMO E1 provide mechanistic insights into SUMO activation and E2 recruitment to E1 , 2005, The EMBO journal.

[8]  C. Pickart,et al.  Mechanisms underlying ubiquitination. , 2001, Annual review of biochemistry.

[9]  H. Saitoh,et al.  Perturbation of SUMOlation Enzyme Ubc9 by Distinct Domain within Nucleoporin RanBP2/Nup358* , 2002, The Journal of Biological Chemistry.

[10]  H. Yasuda,et al.  Involvement of PIAS1 in the sumoylation of tumor suppressor p53. , 2001, Molecular cell.

[11]  M. Scheffner,et al.  A family of proteins structurally and functionally related to the E6-AP ubiquitin-protein ligase. , 1995, Proceedings of the National Academy of Sciences of the United States of America.

[12]  Alexander Varshavsky,et al.  The ubiquitin system. , 1998, Annual review of biochemistry.

[13]  Erica S. Johnson,et al.  An E3-like Factor that Promotes SUMO Conjugation to the Yeast Septins , 2001, Cell.

[14]  R. Deshaies.  SCF and Cullin/Ring H2-based ubiquitin ligases. , 1999, Annual review of cell and developmental biology.

[15]  M. Tatham,et al.  Unique binding interactions among Ubc9, SUMO and RanBP2 reveal a mechanism for SUMO paralog selection , 2005, Nature Structural &Molecular Biology.

[16]  F. Melchior,et al.  A Small Ubiquitin-Related Polypeptide Involved in Targeting RanGAP1 to Nuclear Pore Complex Protein RanBP2 , 1997, Cell.

[17]  M. Kagey,et al.  The Polycomb Protein Pc2 Is a SUMO E3 , 2003, Cell.

[18]  V. Massey,et al.  Determination of dissociation constants and specific rate constants of enzyme-substrate (or protein-ligand) interactions from rapid reaction kinetic data. , 1975, The Journal of biological chemistry.

[19]  C. Ptak,et al.  Structure of a conjugating enzyme-ubiquitin thiolester intermediate reveals a novel role for the ubiquitin tail. , 2001, Structure.

[20]  L. Lois,et al.  STRUCTURES OF THE SMALL UBIQUITIN-LIKE MODIFIER E1 ACTIVATING ENZYME PROVIDE INSIGHTS INTO SUMO ACTIVATION AND THE MECHANISM FOR E2 RECRUITMENT TO E1 , 2005 .

[21]  T. A. Wilkinson,et al.  Identification of a SUMO-binding motif that recognizes SUMO-modified proteins. , 2004, Proceedings of the National Academy of Sciences of the United States of America.

[22]  横山 信彦.  A giant nucleopore protein that binds Ran/TC4 , 1996 .

[23]  G. Blobel,et al.  Nup358, a Cytoplasmically Exposed Nucleoporin with Peptide Repeats, Ran-GTP Binding Sites, Zinc Fingers, a Cyclophilin A Homologous Domain, and a Leucine-rich Region (*) , 1995, The Journal of Biological Chemistry.

[24]  Erica S. Johnson,et al.  Protein modification by SUMO. , 2004, Annual review of biochemistry.

[25]  H. Saitoh,et al.  Enzymes of the SUMO Modification Pathway Localize to Filaments of the Nuclear Pore Complex , 2002, Molecular and Cellular Biology.

[26]  M. Dasso,et al.  The RanGAP1-RanBP2 Complex Is Essential for Microtubule-Kinetochore Interactions In Vivo , 2004, Current Biology.

[27]  Christopher D. Lima,et al.  Structural Basis for E2-Mediated SUMO Conjugation Revealed by a Complex between Ubiquitin-Conjugating Enzyme Ubc9 and RanGAP1 , 2002, Cell.

[28]  K. Sharp,et al.  Protein folding and association: Insights from the interfacial and thermodynamic properties of hydrocarbons , 1991, Proteins.

[29]  P. Howley,et al.  Structure of an E6AP-UbcH7 complex: insights into ubiquitination by the E2-E3 enzyme cascade. , 1999, Science.

[30]  Andrew Emili,et al.  Defining the SUMO-modified Proteome by Multiple Approaches in Saccharomyces cerevisiae* , 2005, Journal of Biological Chemistry.