Mechanism of Lys48‐linked polyubiquitin chain recognition by the Mud1 UBA domain

The ubiquitin‐pathway associated (UBA) domain is a 40‐residue polyubiquitin‐binding motif. The Schizosaccharomyces pombe protein Mud1 is an ortholog of the Saccharomyces cerevisiae DNA‐damage response protein Ddi1 and binds to K48‐linked polyubiquitin through its UBA domain. We have solved the crystal structure of Mud1 UBA at 1.8 Å resolution, revealing a canonical three‐helical UBA fold. We have probed the interactions of this domain using mutagenesis, surface plasmon resonance, NMR and analytical ultracentrifugation. We show that the ubiquitin‐binding surface of Mud1 UBA extends beyond previously recognized motifs and can be functionally dissected into primary and secondary ubiquitin‐binding sites. Mutation of Phe330 to alanine, a residue exposed between helices 2 and 3, significantly reduces the affinity of the Mud1 UBA domain for K48‐linked polyubiquitin, despite leaving the primary binding surface functionally intact. Moreover, K48‐linked diubiquitin binds a single Mud1 UBA domain even in the presence of excess UBA. We therefore propose a mechanism for the recognition of K48‐linked polyubiquitin chains by Mud1 in which diubiquitin units are specifically recognized by a single UBA domain.

[1]  Gottfried Otting,et al.  Alignment of Biological Macromolecules in Novel Nonionic Liquid Crystalline Media for NMR Experiments , 2000 .

[2]  J. Hurley,et al.  A ubiquitin‐binding motif required for intramolecular monoubiquitylation, the CUE domain , 2003, The EMBO journal.

[3]  M. Shirakawa,et al.  Structure of the UBA domain of Dsk2p in complex with ubiquitin molecular determinants for ubiquitin recognition. , 2005, Structure.

[4]  J. Feigon,et al.  Structure of a human DNA repair protein UBA domain that interacts with HIV-1 Vpr , 1998, Nature Structural Biology.

[5]  J. Feigon,et al.  Structural determinants for the binding of ubiquitin‐like domains to the proteasome , 2003, The EMBO journal.

[6]  C. Pickart,et al.  Rad23 Ubiquitin-associated Domains (UBA) Inhibit 26 S Proteasome-catalyzed Proteolysis by Sequestering Lysine 48-linked Polyubiquitin Chains* , 2003, The Journal of Biological Chemistry.

[7]  M D Winn,et al.  An overview of the CCP4 project in protein crystallography: an example of a collaborative project. , 2003, Journal of synchrotron radiation.

[8]  C. Bugg,et al.  Structure of ubiquitin refined at 1.8 A resolution. , 1987, Journal of molecular biology.

[9]  C. Pickart,et al.  Inhibition of the 26 S Proteasome by Polyubiquitin Chains Synthesized to Have Defined Lengths* , 1997, The Journal of Biological Chemistry.

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

[11]  S. Bae,et al.  Binding Surface Mapping of Intra- and Interdomain Interactions among hHR23B, Ubiquitin, and Polyubiquitin Binding Site 2 of S5a* , 2003, Journal of Biological Chemistry.

[12]  Michael Assfalg,et al.  Structural determinants for selective recognition of a Lys48-linked polyubiquitin chain by a UBA domain. , 2005, Molecular cell.

[13]  Hideo Takahashi,et al.  A novel NMR method for determining the interfaces of large protein–protein complexes , 2000, Nature Structural Biology.

[14]  P. Goodford.  A computational procedure for determining energetically favorable binding sites on biologically important macromolecules. , 1985, Journal of medicinal chemistry.

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

[16]  F. Supek,et al.  Ubiquitin Signals Protein Trafficking via Interaction with a Novel Ubiquitin Binding Domain in the Membrane Fusion Regulator, Vps9p , 2003, Current Biology.

[17]  Colin Gordon,et al.  Proteins containing the UBA domain are able to bind to multi-ubiquitin chains , 2001, Nature Cell Biology.

[18]  M. Searle,et al.  Structure of the Ubiquitin-associated Domain of p62 (SQSTM1) and Implications for Mutations That Cause Paget's Disease of Bone* , 2003, Journal of Biological Chemistry.

[19]  J. Feigon,et al.  Solution structures of UBA domains reveal a conserved hydrophobic surface for protein-protein interactions. , 2002, Journal of molecular biology.

[20]  P. Howley,et al.  Ubiquitin recognition by the DNA repair protein hHR23a. , 2003, Biochemistry.

[21]  A. Bax,et al.  Measurement of J and dipolar couplings from simplified two-dimensional NMR spectra. , 1998, Journal of magnetic resonance.

[22]  Walter E. Gall,et al.  Solution structure of the ubiquitin‐binding domain in Swa2p from Saccharomyces cerevisiae , 2004, Proteins: Structure, Function, and Bioinformatics.

[23]  M. Funakoshi,et al.  Budding yeast Dsk2p is a polyubiquitin-binding protein that can interact with the proteasome , 2002, Proceedings of the National Academy of Sciences of the United States of America.

[24]  N. Williams.  Cautious welcome for GM crops , 2001, Current Biology.

[25]  George M Sheldrick,et al.  Substructure solution with SHELXD. , 2002, Acta crystallographica. Section D, Biological crystallography.

[26]  Q. Deveraux,et al.  A 26 S protease subunit that binds ubiquitin conjugates. , 1994, The Journal of biological chemistry.

[27]  D. M. Krylov,et al.  Correspondence A novel family of predicted retroviral-like aspartyl proteases with a possible key role in eukaryotic cell cycle control , 2001, Current Biology.

[28]  W. Salerno,et al.  Solution Structure of a CUE-Ubiquitin Complex Reveals a Conserved Mode of Ubiquitin Binding , 2003, Cell.

[29]  P. Bucher,et al.  The UBA domain: a sequence motif present in multiple enzyme classes of the ubiquitination pathway. , 1996, Trends in biochemical sciences.

[30]  J. Feigon,et al.  Specificity of the Interaction between Ubiquitin-associated Domains and Ubiquitin* , 2004, Journal of Biological Chemistry.

[31]  G. Bricogne,et al.  [27] Maximum-likelihood heavy-atom parameter refinement for multiple isomorphous replacement and multiwavelength anomalous diffraction methods. , 1997, Methods in enzymology.

[32]  M. Carson,et al.  Structure of a diubiquitin conjugate and a model for interaction with ubiquitin conjugating enzyme (E2). , 1993, The Journal of biological chemistry.

[33]  C. Pickart,et al.  Ubiquitin in chains. , 2000, Trends in biochemical sciences.

[34]  C. Pickart,et al.  Binding of polyubiquitin chains to ubiquitin-associated (UBA) domains of HHR23A. , 2004, Journal of molecular biology.

[35]  G. Sheldrick,et al.  Locating the anomalous scatterer substructures in halide and sulfur phasing. , 2003, Acta crystallographica. Section D, Biological crystallography.

[36]  W. Dalton,et al.  The proteasome. , 2004, Seminars in oncology.

[37]  N. Krishna,et al.  Sequestosome 1/p62 Is a Polyubiquitin Chain Binding Protein Involved in Ubiquitin Proteasome Degradation , 2004, Molecular and Cellular Biology.

[38]  C. Pickart,et al.  A 25-kilodalton ubiquitin carrier protein (E2) catalyzes multi-ubiquitin chain synthesis via lysine 48 of ubiquitin. , 1990, The Journal of biological chemistry.

[39]  J. Strominger,et al.  p62, a Phosphotyrosine-independent Ligand of the SH2 Domain of p56lck, Belongs to a New Class of Ubiquitin-binding Proteins* , 1996, The Journal of Biological Chemistry.

[40]  D. J. Clarke,et al.  UBA domains mediate protein-protein interactions between two DNA damage-inducible proteins. , 2001, Journal of molecular biology.

[41]  D. J. Clarke,et al.  UBA domains of DNA damage-inducible proteins interact with ubiquitin , 2001, Nature Structural Biology.

[42]  D. Fushman,et al.  Structural properties of polyubiquitin chains in solution. , 2002, Journal of molecular biology.

[43]  J. Hurley,et al.  Mechanism of Ubiquitin Recognition by the CUE Domain of Vps9p , 2003, Cell.

[44]  Li Chen,et al.  Rad23 Promotes the Targeting of Proteolytic Substrates to the Proteasome , 2002, Molecular and Cellular Biology.

[45]  Aydin Haririnia,et al.  Solution Conformation of Lys63-linked Di-ubiquitin Chain Provides Clues to Functional Diversity of Polyubiquitin Signaling* , 2004, Journal of Biological Chemistry.

[46]  P. Howley,et al.  DNA-repair protein hHR23a alters its protein structure upon binding proteasomal subunit S5a , 2003, Proceedings of the National Academy of Sciences of the United States of America.

[47]  J. Hus,et al.  A novel interactive tool for rigid-body modeling of multi-domain macromolecules using residual dipolar couplings , 2001, Journal of biomolecular NMR.

[48]  George M Church,et al.  Localization to the Proteasome Is Sufficient for Degradation* , 2004, Journal of Biological Chemistry.

[49]  D. Finley,et al.  Rad23 and Rpn10 Serve as Alternative Ubiquitin Receptors for the Proteasome* , 2004, Journal of Biological Chemistry.