Phosphorylation releases constraints to domain motion in ERK2.
Phosphorylation releases constraints to domain motion in ERK2.
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磷酸化释放了 ERK2 结构域运动的限制。
DOI：
10.1073/pnas.1318899111
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发表时间：
2014
影响因子：
11.1
通讯作者：
Ahn,NatalieG
中科院分区：
文献类型：
--
作者：
Xiao,Yao;Lee,Thomas;Latham,MichaelParker;Warner,LisaRose;Tanimoto,Akiko;Pardi,Arthur;Ahn,NatalieG
蛋白质运动通过不完全理解的机制控制酶催化。在此，NMR 13 C弛豫分散实验用于监测响应于MAP激酶ERK 2的磷酸化活化而发生的侧链运动的变化。Ile、Leu和瓦尔残基上甲基侧链的NMR数据显示了ERK 2不同活性状态之间在微秒至毫秒时间范围内的构象交换动力学变化。在失活的，未磷酸化的ERK 2，局部构象交换甲基侧链之间观察到，几乎没有证据表明残基之间的耦合。在MAP激酶激酶1的双重磷酸化后，ERK 2中指定甲基的动力学在整个保守激酶核心中发生改变，包括催化口袋中的许多残基。活性ERK 2中的大多数残基符合单一的构象交换过程，kex = 300 s−1（kAB = 240 s−1/kBA = 60 s−1）和pA/pB = 20%/80%，表明涉及两种状态之间相互转换的全局结构域运动。ERK 2的一个突变体，被设计为增强连接N-和C-末端结构域的铰链区的构象移动性，也诱导了整个激酶核心的双态构象交换，交换特性为kex ≥ 500 s-1（kAB ≥ 15 s-1/kBA ≥ 485 s-1）和pA/pB ≥ 97%/3%。因此，ERK 2的磷酸化和激活导致构象交换动力学的急剧变化，可能是通过释放铰链处的约束。
Protein motions control enzyme catalysis through mechanisms that are incompletely understood. Here NMR13C relaxation dispersion experiments were used to monitor changes in side-chain motions that occur in response to activation by phosphorylation of the MAP kinase ERK2. NMR data for the methyl side chains on Ile, Leu, and Val residues showed changes in conformational exchange dynamics in the microsecond-to-millisecond time regime between the different activity states of ERK2. In inactive, unphosphorylated ERK2, localized conformational exchange was observed among methyl side chains, with little evidence for coupling between residues. Upon dual phosphorylation by MAP kinase kinase 1, the dynamics of assigned methyls in ERK2 were altered throughout the conserved kinase core, including many residues in the catalytic pocket. The majority of residues in active ERK2 fit to a single conformational exchange process, withkex≈ 300 s−1(kAB≈ 240 s−1/kBA≈ 60 s−1) and pA/pB≈ 20%/80%, suggesting global domain motions involving interconversion between two states. A mutant of ERK2, engineered to enhance conformational mobility at the hinge region linking the N- and C-terminal domains, also induced two-state conformational exchange throughout the kinase core, with exchange properties ofkex≈ 500 s−1(kAB≈ 15 s−1/kBA≈ 485 s−1) and pA/pB≈ 97%/3%. Thus, phosphorylation and activation of ERK2 lead to a dramatic shift in conformational exchange dynamics, likely through release of constraints at the hinge.