Yunus Ali A, Lima Christopher D
Structural Biology Program, Sloan-Kettering Institute, New York, New York 10021, USA.
Nat Struct Mol Biol. 2006 Jun;13(6):491-9. doi: 10.1038/nsmb1104. Epub 2006 May 28.
E2 conjugating proteins that transfer ubiquitin and ubiquitin-like modifiers to substrate lysine residues must first activate the lysine nucleophile for conjugation. Genetic complementation revealed three side chains of the E2 Ubc9 that were crucial for normal growth. Kinetic analysis revealed modest binding defects but substantially lowered catalytic rates for these mutant alleles with respect to wild-type Ubc9. X-ray structures for wild-type and mutant human Ubc9-RanGAP1 complexes showed partial loss of contacts to the substrate lysine in mutant complexes. Computational analysis predicted pK perturbations for the substrate lysine, and Ubc9 mutations weakened pK suppression through improper side chain coordination. Biochemical studies with p53, RanGAP1 and the Nup358/RanBP2 E3 were used to determine rate constants and pK values, confirming both structural and computational predictions. It seems that Ubc9 uses an indirect mechanism to activate lysine for conjugation that may be conserved among E2 family members.
将泛素和泛素样修饰物转移至底物赖氨酸残基的E2缀合蛋白必须首先激活赖氨酸亲核试剂以进行缀合。遗传互补揭示了E2 Ubc9的三个侧链对正常生长至关重要。动力学分析显示结合缺陷适度，但相对于野生型Ubc9，这些突变等位基因的催化速率大幅降低。野生型和突变型人Ubc9-RanGAP1复合物的X射线结构显示突变复合物中与底物赖氨酸的接触部分丧失。计算分析预测了底物赖氨酸的pK扰动，并且Ubc9突变通过不适当的侧链配位削弱了pK抑制。使用p53、RanGAP1和Nup358/RanBP2 E3进行的生化研究用于确定速率常数和pK值，证实了结构和计算预测。似乎Ubc9使用一种间接机制来激活赖氨酸以进行缀合，这种机制可能在E2家族成员中保守。
