title: Activation of UbcH5c~Ub is the result of a shift in interdomain motions of the conjugate bound to U-box E3 ligase E4B.
abstract: Post-translational modification of proteins with ubiquitin is mediated by dynamic multienzyme machinery (E1, E2, and E3). E3 ubiquitin ligases play a key role acting as both scaffolds to bring reactants together and activators to catalyze ubiquitin (Ub) transfer from E2~Ub conjugates to substrates. Our recent studies provided insights into the mechanism of the activation event; binding of an E3 to an E2~Ub conjugate was found to affect the motions of E2~Ub and allosterically stimulate Ub transfer. This proposed mechanism implies that the dynamics of the conjugate, which has been shown to occupy a wide range of E2~Ub orientations, will be altered significantly upon binding of E3. To directly assess the effect of E3 binding on E2~Ub dynamics, we undertook an in-depth comparative analysis of (15)N nuclear magnetic resonance relaxation of UbcH5c~Ub in the absence and presence of the E3 ligase, E4B. Challenges encountered in deciphering interdomain motions for this ternary complex are discussed along with the limitations of the current approaches. Notably, although a reduction in interdomain dynamics of UbcH5c~Ub is observed upon binding to E4B, Ub retains an extensive degree of flexibility. These results provide strong support for our dynamic model of a significant orientational bias of Ub toward a more closed conformation in the E3/E2~Ub complex.
authors.affiliation: Department of Biochemistry, Center for Structural Biology, Vanderbilt University, Nashville, TN 37232, USA.
authors.forename: Sarah E
authors.initials: SE
authors.lastname: Soss
authors.forename: Rachel E
authors.initials: RE
authors.lastname: Klevit
authors.forename: Walter J
authors.initials: WJ
authors.lastname: Chazin
mesh_terms: D055162:Biocatalysis; D008958:Models, Molecular; D019906:Nuclear Magnetic Resonance, Biomolecular; D011485:Protein Binding; D044763:Ubiquitin-Conjugating Enzymes / Q000378:metabolism*; D044767:Ubiquitin-Protein Ligases / Q000378:metabolism*
chemical_list: C097310:UBE2D3 protein, human; D044763:Ubiquitin-Conjugating Enzymes; D044767:Ubiquitin-Protein Ligases
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