Activation of UbcH5cUb Is the Result of a Shift in Interdomain Motions of the Conjugate Bound to U-Box E3 Ligase E4B
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- 作者：Sarah E. Soss ; Rachel E. Klevit ; Walter J. Chazin
- 刊名：Biochemistry
- 出版年：2013
- 出版时间：April 30, 2013
- 年：2013
- 卷：52
- 期：17
- 页码：2991-2999
- 全文大小：380K
- 年卷期：v.52,no.17(April 30, 2013)
- ISSN：1520-4995
文摘
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 E2Ub conjugates to substrates. Our recent studies provided insights into the mechanism of the activation event; binding of an E3 to an E2Ub conjugate was found to affect the motions of E2Ub 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 E2Ub orientations, will be altered significantly upon binding of E3. To directly assess the effect of E3 binding on E2Ub dynamics, we undertook an in-depth comparative analysis of 15N nuclear magnetic resonance relaxation of UbcH5cUb 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 UbcH5cUb 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/E2Ub complex.