Structure and analysis of a complex between SUMO and Ubc9 illustrates features of a conserved E2-Ubl interaction.

The SUMO E2 Ubc9 serves as a lynchpin in the SUMO conjugation pathway, interacting with the SUMO E1 during activation, with thioester linked SUMO after E1 transfer and with the substrate and SUMO E3 ligases during conjugation. Here, we describe the structure determination of a non-covalent complex between human Ubc9 and SUMO-1 at 2.4 A resolution. Non-covalent interactions between Ubc9 and SUMO are conserved in human and yeast insomuch as human Ubc9 interacts with each of the human SUMO isoforms, and yeast Ubc9 interacts with Smt3, the yeast SUMO ortholog. Structural comparisons reveal similarities to several other non-covalent complexes in the ubiquitin pathway, suggesting that the non-covalent Ubc9-SUMO interface may be important for poly-SUMO chain formation, for E2 recruitment to SUMO conjugated substrates, or for mediating E2 interactions with either E1 or E3 ligases. Biochemical analysis suggests that this surface is less important for E1 activation or di-SUMO-2 formation, but more important for E3 interactions and for poly-SUMO chain formation when the chain exceeds more than two SUMO proteins.

[1]  Fumio Hanaoka,et al.  Crystal structure of thymine DNA glycosylase conjugated to SUMO-1 , 2005, Nature.

[2]  David Reverter,et al.  Structural basis for SENP2 protease interactions with SUMO precursors and conjugated substrates , 2006, Nature Structural &Molecular Biology.

[3]  Bernhard Kuster,et al.  A Proteome-wide Approach Identifies Sumoylated Substrate Proteins in Yeast* , 2004, Journal of Biological Chemistry.

[4]  F. Melchior,et al.  SUMO: ligases, isopeptidases and nuclear pores. , 2003, Trends in biochemical sciences.

[5]  J. Zou,et al.  Improved methods for building protein models in electron density maps and the location of errors in these models. , 1991, Acta crystallographica. Section A, Foundations of crystallography.

[6]  M. Tatham,et al.  Polymeric Chains of SUMO-2 and SUMO-3 Are Conjugated to Protein Substrates by SAE1/SAE2 and Ubc9* , 2001, The Journal of Biological Chemistry.

[7]  M. Hochstrasser,et al.  Modification of proteins by ubiquitin and ubiquitin-like proteins. , 2006, Annual review of cell and developmental biology.

[8]  Z. Otwinowski,et al.  [20] Processing of X-ray diffraction data collected in oscillation mode. , 1997, Methods in enzymology.

[9]  A Perrakis,et al.  Crystal Structure of Murine/Human Ubc9 Provides Insight into the Variability of the Ubiquitin-conjugating System* , 1997, The Journal of Biological Chemistry.

[10]  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.

[11]  M. Bjornsti,et al.  Structure of a SUMO-binding-motif mimic bound to Smt3p-Ubc9p: conservation of a non-covalent ubiquitin-like protein-E2 complex as a platform for selective interactions within a SUMO pathway. , 2007, Journal of molecular biology.

[12]  C. Slaughter,et al.  Identification of a Multifunctional Binding Site on Ubc9p Required for Smt3p Conjugation* , 2002, The Journal of Biological Chemistry.

[13]  Erica S. Johnson,et al.  The SUMO Isopeptidase Ulp2 Prevents Accumulation of SUMO Chains in Yeast* , 2003, Journal of Biological Chemistry.

[14]  W. Delano.  The PyMOL Molecular Graphics System , 2002 .

[15]  A. Ciechanover,et al.  The ubiquitin system. , 1998, Annual review of biochemistry.

[16]  David J. Chen,et al.  The Binding Interface between an E2 (UBC9) and a Ubiquitin Homologue (UBL1)* , 1999, The Journal of Biological Chemistry.

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

[18]  M. White,et al.  Broad Spectrum Identification of Cellular Small Ubiquitin-related Modifier (SUMO) Substrate Proteins* , 2004, Journal of Biological Chemistry.

[19]  H. Senn,et al.  Characterization of the binding interface between ubiquitin and class I human ubiquitin-conjugating enzyme 2b by multidimensional heteronuclear NMR spectroscopy in solution. , 1999, Journal of molecular biology.

[20]  J. Naismith,et al.  Structure of ubiquitin-conjugating enzyme 9 displays significant differences with other ubiquitin-conjugating enzymes which may reflect its specificity for sumo rather than ubiquitin. , 1998, Acta crystallographica. Section D, Biological crystallography.

[21]  Huilin Zhou,et al.  Global Analyses of Sumoylated Proteins in Saccharomyces cerevisiae , 2004, Journal of Biological Chemistry.

[22]  R J Read,et al.  Crystallography & NMR system: A new software suite for macromolecular structure determination. , 1998, Acta crystallographica. Section D, Biological crystallography.

[23]  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.

[24]  J. Holton,et al.  Basis for a ubiquitin-like protein thioester switch toggling E1–E2 affinity , 2007, Nature.

[25]  M. Hochstrasser,et al.  Substrate Targeting in the Ubiquitin System , 1999, Cell.

[26]  Collaborative Computational,et al.  The CCP4 suite: programs for protein crystallography. , 1994, Acta crystallographica. Section D, Biological crystallography.

[27]  J. Thornton,et al.  PROCHECK: a program to check the stereochemical quality of protein structures , 1993 .

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

[29]  Y. Saeki,et al.  Direct interactions between NEDD8 and ubiquitin E2 conjugating enzymes upregulate cullin-based E3 ligase activity , 2007, Nature Structural &Molecular Biology.

[30]  Cynthia Wolberger,et al.  Mms2–Ubc13 covalently bound to ubiquitin reveals the structural basis of linkage-specific polyubiquitin chain formation , 2006, Nature Structural &Molecular Biology.

[31]  C. Pickart,et al.  Molecular Insights into Polyubiquitin Chain Assembly Crystal Structure of the Mms2/Ubc13 Heterodimer , 2001, Cell.

[32]  D. Hoyt,et al.  A UbcH5/ubiquitin noncovalent complex is required for processive BRCA1-directed ubiquitination. , 2006, Molecular cell.

[33]  C. Lima,et al.  Lysine activation and functional analysis of E2-mediated conjugation in the SUMO pathway , 2006, Nature Structural &Molecular Biology.

[34]  John R Yates,et al.  Global Analysis of Protein Sumoylation in Saccharomyces cerevisiae* , 2004, Journal of Biological Chemistry.

[35]  Chia-Hui Yeh,et al.  SUMO modifications control assembly of synaptonemal complex and polycomplex in meiosis of Saccharomyces cerevisiae. , 2006, Genes & development.

[36]  C. Lima,et al.  Purification and activity assays for Ubc9, the ubiquitin-conjugating enzyme for the small ubiquitin-like modifier SUMO. , 2005, Methods in enzymology.

[37]  David Owerbach,et al.  A proline-90 residue unique to SUMO-4 prevents maturation and sumoylation. , 2005, Biochemical and biophysical research communications.

[38]  David Reverter,et al.  A basis for SUMO protease specificity provided by analysis of human Senp2 and a Senp2-SUMO complex. , 2004, Structure.

[39]  M. Hochstrasser.  There's the rub: a novel ubiquitin-like modification linked to cell cycle regulation. , 1998, Genes & development.

[40]  David Reverter,et al.  Insights into E3 ligase activity revealed by a SUMO–RanGAP1–Ubc9–Nup358 complex , 2005, Nature.

[41]  David C Schwartz,et al.  A superfamily of protein tags: ubiquitin, SUMO and related modifiers. , 2003, Trends in biochemical sciences.

[42]  M. Tatham,et al.  Role of an N-terminal site of Ubc9 in SUMO-1, -2, and -3 binding and conjugation. , 2003, Biochemistry.

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