title: Structural and biochemical studies of the open state of Lys48-linked diubiquitin.
abstract: Ubiquitin (Ub) is a small protein highly conserved among eukaryotes and involved in practically all aspects of eukaryotic cell biology. Polymeric chains assembled from covalently-linked Ub monomers function as molecular signals in the regulation of a host of cellular processes. Our previous studies have shown that the predominant state of Lys48-linked di- and tetra-Ub chains at near-physiological conditions is a closed conformation, in which the Ub-Ub interface is formed by the hydrophobic surface residues of the adjacent Ub units. Because these very residues are involved in (poly)Ub interactions with the majority of Ub-binding proteins, their sequestration at the Ub-Ub interface renders the closed conformation of polyUb binding incompetent. Thus the existence of open conformation(s) and the interdomain motions opening and closing the Ub-Ub interface is critical for the recognition of Lys48-linked polyUb by its receptors. Knowledge of the conformational properties of a polyUb signal is essential for our understanding of its specific recognition by various Ub-receptors. Despite their functional importance, open states of Lys48-linked chains are poorly characterized. Here we report a crystal structure of the open state of Lys48-linked di-Ub. Moreover, using NMR, we examined interactions of the open state of this chain (at pH4.5) with a Lys48-linkage-selective receptor, the UBA2 domain of a shuttle protein hHR23a. Our results show that di-Ub binds UBA2 in the same mode and with comparable affinity as the closed state. Our data suggest a mechanism for polyUb signal recognition, whereby Ub-binding proteins select specific conformations out of the available ensemble of polyUb chain conformations. This article is part of a Special Issue entitled: Ubiquitin Drug Discovery and Diagnostics.
authors.affiliation: Department of Chemistry and Biochemistry, University of Maryland, College Park, MD 20742, USA.
authors.forename: Ming-Yih
authors.initials: MY
authors.lastname: Lai
authors.forename: Daoning
authors.initials: D
authors.lastname: Zhang
authors.forename: Nicole
authors.initials: N
authors.lastname: Laronde-Leblanc
authors.forename: David
authors.initials: D
authors.lastname: Fushman
mesh_terms: D018360:Crystallography, X-Ray; D045643:DNA Repair Enzymes / Q000737:chemistry* / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000737:chemistry* / Q000378:metabolism; D006801:Humans; D008239:Lysine / Q000737:chemistry* / Q000378:metabolism; D009682:Magnetic Resonance Spectroscopy; D008958:Models, Molecular; D025821:Polyubiquitin / Q000737:chemistry* / Q000378:metabolism; D011485:Protein Binding; D011487:Protein Conformation; D044764:Ubiquitin-Activating Enzymes / Q000737:chemistry* / Q000378:metabolism
chemical_list: D004268:DNA-Binding Proteins; C470301:UBA2 protein, human; D025821:Polyubiquitin; C086985:RAD23A protein, human; D044764:Ubiquitin-Activating Enzymes; D045643:DNA Repair Enzymes; D008239:Lysine