title: Mechanism of polyubiquitin chain recognition by the human ubiquitin conjugating enzyme Ube2g2.
abstract: Ube2g2 is a human ubiquitin conjugating (E2) enzyme involved in the endoplasmic reticulum-associated degradation pathway, which is responsible for the identification and degradation of unfolded and misfolded proteins in the endoplasmic reticulum compartment. The Ube2g2-specific role is the assembly of Lys-48-linked polyubiquitin chains, which constitutes a signal for proteasomal degradation when attached to a substrate protein. NMR chemical shift perturbation and paramagnetic relaxation enhancement approaches were employed to characterize the binding interaction between Ube2g2 and ubiquitin, Lys-48-linked diubiquitin, and Lys-63-linked diubiquitin. Results demonstrate that ubiquitin binds to Ube2g2 with an affinity of 90 μM in two different orientations that are rotated by 180° in models generated by the RosettaDock modeling suite. The binding of Ube2g2 to Lys-48- and Lys-63-linked diubiquitin is primarily driven by interactions with individual ubiquitin subunits, with a clear preference for the subunit containing the free Lys-48 or Lys-63 side chain (i.e. the distal subunit). This preference is particularly striking in the case of Lys-48-linked diubiquitin, which exhibits an ∼3-fold difference in affinities between the two ubiquitin subunits. This difference can be attributed to the partial steric occlusion of the subunit whose Lys-48 side chain is involved in the isopeptide linkage. As such, these results suggest that Lys-48-linked polyubiquitin chains may be designed to bind certain proteins like Ube2g2 such that the terminal ubiquitin subunit carrying the reactive Lys-48 side chain can be positioned properly for chain elongation regardless of chain length.
authors.affiliation: Department of Chemistry, Johns Hopkins University, Baltimore, Maryland 21218, USA.
authors.forename: William E
authors.initials: WE
authors.lastname: Bocik
authors.forename: Aroop
authors.initials: A
authors.lastname: Sircar
authors.forename: Jeffrey J
authors.initials: JJ
authors.lastname: Gray
authors.forename: Joel R
authors.initials: JR
authors.lastname: Tolman
mesh_terms: D006801:Humans; D008239:Lysine; D009682:Magnetic Resonance Spectroscopy; D008958:Models, Molecular; D025821:Polyubiquitin / Q000378:metabolism*; D011485:Protein Binding; D025801:Ubiquitin / Q000378:metabolism; D044763:Ubiquitin-Conjugating Enzymes / Q000378:metabolism*
chemical_list: D025801:Ubiquitin; D025821:Polyubiquitin; C471617:UBE2G2 protein, human; D044763:Ubiquitin-Conjugating Enzymes; D008239:Lysine