The intrinsic affinity between E2 and the Cys domain of E1 in ubiquitin-like modifications.

Ubiquitin-like modifications, which are carried out by similar biochemical mechanisms, regulate nearly every aspect of cellular function. Despite the recent advancements in characterizing their enzymology, our knowledge about the dynamic processes of these modifications is still fragmentary. In this study, we have uncovered an intrinsic affinity between the SUMO E2 and the Cys domain of SUMO E1. NMR studies in combination with paramagnetic spin labeling demonstrate that this interaction is mediated by previously unknown interfaces on both E1 and E2 and places the two catalytic Cys residues of the two enzymes in close proximity. Site-directed mutagenesis and enzymatic assays indicate that the interaction is fundamentally important for the transfer of SUMO from E1 to E2. Results from this study suggest that the interaction between E2 and the Cys domain of E1 participates in guiding the E2's translocation to E1's enzymatic active site in ubiquitin-like modifications.

[1]  C. Dominguez,et al.  HADDOCK: a protein-protein docking approach based on biochemical or biophysical information. , 2003, Journal of the American Chemical Society.

[2]  A. Haas,et al.  Protein Interactions within the N-end Rule Ubiquitin Ligation Pathway* , 2003, The Journal of Biological Chemistry.

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

[4]  T. Blundell,et al.  Comparative protein modelling by satisfaction of spatial restraints. , 1993, Journal of molecular biology.

[5]  Pier Paolo Pandolfi,et al.  The mechanisms of PML-nuclear body formation. , 2006, Molecular cell.

[6]  R. Wynn,et al.  Steady-State Kinetic Analysis of Human Ubiquitin-Activating Enzyme (E1) Using a Fluorescently Labeled Ubiquitin Substrate , 2000, Journal of protein chemistry.

[7]  S. Grzesiek,et al.  NMRPipe: A multidimensional spectral processing system based on UNIX pipes , 1995, Journal of biomolecular NMR.

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

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

[10]  A. Bax,et al.  Protein backbone angle restraints from searching a database for chemical shift and sequence homology , 1999, Journal of biomolecular NMR.

[11]  D. Shortle,et al.  Characterization of long-range structure in the denatured state of staphylococcal nuclease. II. Distance restraints from paramagnetic relaxation and calculation of an ensemble of structures. , 1997, Journal of molecular biology.

[12]  A. Varshavsky,et al.  The ubiquitin system. , 1997, Trends in biochemical sciences.

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

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

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

[16]  Donghai Lin,et al.  Identification of a Substrate Recognition Site on Ubc9* , 2002, The Journal of Biological Chemistry.

[17]  B. Schulman,et al.  Insights into the ubiquitin transfer cascade from the structure of the activating enzyme for NEDD8 , 2003, Nature.

[18]  A. Haas,et al.  Conservation in the Mechanism of Nedd8 Activation by the Human AppBp1-Uba3 Heterodimer* , 2003, Journal of Biological Chemistry.

[19]  K. Wilkinson.  Regulation of ubiquitin‐dependent processes by deubiquitinating enzymes , 1997, FASEB journal : official publication of the Federation of American Societies for Experimental Biology.

[20]  T. Krugh.  9 – Spin-Label-Induced Nuclear Magnetic Resonance Relaxation Studies of Enzymes , 1976 .

[21]  A. Haas,et al.  Pathways of ubiquitin conjugation , 1997, FASEB journal : official publication of the Federation of American Societies for Experimental Biology.

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

[23]  G. Wagner,et al.  Utilization of site-directed spin labeling and high-resolution heteronuclear nuclear magnetic resonance for global fold determination of large proteins with limited nuclear overhauser effect data. , 2000, Biochemistry.

[24]  I. Rose.  Ubiquitin at Fox Chase. , 2005, Cell death and differentiation.

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

[26]  Y. Yuan,et al.  Conformational flexibility of a ubiquitin conjugation enzyme (E2). , 1999, Biochemistry.

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

[28]  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.

[29]  Rebecca L Rich,et al.  Structure and functional interactions of the Tsg101 UEV domain , 2002, The EMBO journal.

[30]  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.

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