Catalytic Domain Dynamics in Protein Kinases

许多人类癌症的进展与高水平的表达和催化活性相关。 酪氨酸激酶（SFK）的Src家族的活性使其成为关键的治疗靶标。即使 活性和非活性形式的SFK催化结构域的多种晶体结构的可用性， 对其催化调节的理解是不可用的。公认的导致大幅度增加的核心步骤 在催化活性中，最重要的是“活化环”中调节性酪氨酸残基（Tyract）的磷酸化。这 化学改性被认为会引起局部和远程相互作用的变化， 催化领域内的调节动力学。虽然其中一些变化是从晶体中推断出来的， 结构，缺乏直接证据。溶液核磁共振，最适合解决这个问题的生物物理方法， 以前由于细菌表达和纯化足量可溶性， 适当折叠的蛋白质，用于用NMR活性同位素进行经济上可行的标记。我们通过选择 优化的构建体，与分子伴侣的共表达和纯化方案的优化，实现了 细菌产生大量同位素标记的c-Src催化结构域的能力， 原型SFK及其Tyract磷酸化形式。这一点，再加上超高场的可用性 NMR仪器（900 MHz）配备最新一代低温探头和高质量的 最初的NMR光谱，使详细的NMR研究的催化结构域的SFKs可行的第一 时间我们将利用新的NMR方法来充分表征c-Src催化结构域的动力学， 它们对Tyract磷酸化的修饰，它们对酶活性调节的影响， 它们被三种特定类型的小分子抑制剂中的每一种干扰的机制。 SFK使用额外的非催化结构域来调节催化活性，而其他蛋白激酶 例如细胞外信号调节激酶（ERK）类的丝氨酸/苏氨酸激酶 催化域本身代替外部域。值得注意的是，整体结构和关键监管 催化结构域的元件在蛋白激酶中是高度保守的。因此，预计某些 功能动力学的模式将是保守的，而其他模式将根据激酶的类别而变化。 我们将通过确定ERK 2（一种典型的ERK）的功能动力学， 在正调节激活环Thr-X-Tyr基序的双重磷酸化后的修饰，用于比较 关于C-Src我们还将研究对接相互作用（目前在SFK中未确定）的修饰效应。 与调节磷酸酶，在ERK 2的功能动力学。 了解激酶激活的动态基础将可能允许改善 用于干预激酶相关病症的新治疗剂的当前和开发， 特别是在癌症和自身免疫疾病中。

Progression of a host of human cancers is associated with elevated levels of expression and catalytic activity of the Src family of tyrosine kinases (SFKs) making them key therapeutic targets. Even with the availability of multiple crystal structures of active and inactive forms of the SFK catalytic domain, a complete understanding of its catalytic regulation is unavailable. A central step recognized to lead to a dramatic increase in catalytic activity is the phosphorylation of a regulatory tyrosine residue (Tyract) in the "activation loop". This chemical modification is presumed to cause changes in local and long-range interactions and modification of the regulatory dynamics within the catalytic domain. Though some of these changes are inferred from crystal structures, direct evidence is lacking. Solution NMR, the biophysical method best suited to tackle this problem, was previously hindered by difficulties in bacterial expression and purification of sufficient quantities of soluble, properly folded protein for economically viable labeling with NMR-active isotopes. We have through a choice of optimal constructs, co-expression with chaperones and optimization of the purification protocol, achieved the ability to bacterially produce large quantities of the isotopically-labeled catalytic domain of c-Src, the prototypical SFK, and of its Tyract phosphorylated form. This, together with the availability of ultra-high field NMR instrumentation (900 MHz) equipped with the latest generation cryogenic probes and the high-quality of the initial NMR spectra, make the detailed NMR studies of the catalytic domain of the SFKs viable for the first time. We will utilize novel NMR methodology to fully characterize the dynamics of the c-Src catalytic domain, their modifications upon Tyract phosphorylation, their influence on the regulation of enzymatic activity and the mechanism of their perturbation by each of three specific classes of small molecule inhibitors. The SFKs use additional non-catalytic domains to modulate catalytic activity while other protein kinases such as the extracellular signal-regulated kinase (ERK) class of serine/threonine kinases use insertions within the catalytic domain itself in lieu of external domains. Notably, the overall structure and key regulatory elements of the catalytic domain are highly conserved amongst protein kinases. It is thus expected that certain modes of functional dynamics would be conserved while others would vary depending on the class of kinase. We will investigate these effects by ascertaining the functional dynamics in ERK2 (a prototypical ERK), their modification upon dual-phosphorylation of a positive-regulatory activation-loop Thr-X-Tyr motif, for comparison with c-Src. We will also investigate the modifying effects of docking interactions (currently unidentified in SFKs) with regulatory phosphatases, on the functional dynamics in ERK2. Understanding the dynamic underpinnings of kinase activation will likely permit the improvement of current, and the development of new, therapeutic agents for intervention in kinase-associated disorders, especially in cancer and auto-immune diseases.