Mapping intersubunit interactions of the regulatory subunit (RIα) in the type I holoenzyme of protein kinase A by amide hydrogen/deuterium exchange mass spectrometry (DXMS)
Mapping intersubunit interactions of the regulatory subunit (RIα) in the type I holoenzyme of protein kinase A by amide hydrogen/deuterium exchange mass spectrometry (DXMS)
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DOI：
10.1016/j.jmb.2004.05.042
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发表时间：
2004-07-23
影响因子：
5.6
通讯作者：
Woods, VL
中科院分区：
文献类型：
--
作者：
Hamuro, Y;Anand, GS;Woods, VL
蛋白激酶A （PKA）是细胞中cAMP信号传导的中心位点，由调控亚基(R)和催化亚基(C)组成。c -亚基通过在四聚体全酶复合体（R2C2）中与r -亚基二聚体结合而保持无活性状态。PKA被cAMP与r亚基结合而激活，从而引起构象变化，导致活性c亚基释放。因此，c -亚基的酶活性由cAMP通过r -亚基调节，r -亚基在cAMP和c -亚基结合状态之间切换。r -亚基由二聚化/时钟（D/D）结构域组成，连接两个cAMP结合结构域（cAMP: a和cAMP:B）。虽然已知r -亚基缺失突变体的游离c亚基和camp结合状态的晶体结构，但没有全酶复合物的结构或r -亚基的无camp状态。这是理解的重要一步。cAMP依赖的PKA激活是为了绘制R-C界面，并表征r亚基与cAMP和c亚基的互斥相互作用。酰胺氢/氘交换质谱法是一种合适的方法，它提供了对溶液中r亚基不同状态的见解，从而可以绘制cAMP和c亚基对r亚基不同区域的影响。我们的研究将c -亚基的相互作用定位在cAMP: a结构域和连接子区域的一个小的连续表面上。此外，c -亚基结合导致两个cAMP结构域内酰胺氢交换增加，这表明这些区域在全酶中变得更加灵活，并准备与cAMP结合。此外，RIalpha和先前研究的RIIbeta对camp结合的保护模式的差异表明，camp依赖的PKA活性调节存在同种异构体特异性差异。(C) 2004 Elsevier Ltd.版权所有。
Protein kinase A (PKA), a central locus for cAMP signaling in the cell, is composed of regulatory (R) and catalytic (C) subunits. The C-subunits are maintained in an inactive state by binding to the R-subunit dimer in a tetrameric holoenzyme complex (R2C2). PKA is activated by cAMP binding to the R-subunits which induces a conformational change leading to release of the active C-subunit. Enzymatic activity of the C-subunit is thus regulated by cAMP via the R-subunit, which toggles between cAMP and C-subunit bound states. The R-subunit is composed of a dimerization/clocking (D/D) domain connected to two cAMP-binding domains (cAMP:A and cAMP:B). While crystal structures of the free C-subunit and cAMP-bound states of a deletion mutant of the R-subunit are known, there is no structure of the holoenzyme complex or of the cAMP-free state of the R-subunit. An important step in understanding the. cAMP-dependent activation of PKA is to map the R-C interface and, characterize the mutually exclusive interactions of the R-subunit with cAMP and C-subunit. Amide hydrogen/deuterium exchange mass spectrometry is a suitable method that has provided insights into the different states of the R-subunit in solution, thereby allowing mapping of the effects of cAMP and C-subunit on different regions of the R-subunit. Our study has localized interactions with the C-subunit to a small contiguous surface on the cAMP:A domain and the linker region. In addition, C-subunit binding causes increased amide hydrogen exchange within both cAMP-domains, suggesting that these regions become more flexible in the holoenzyme and are primed to bind cAMP. Furthermore, the difference in the protection patterns between RIalpha and the previously studied RIIbeta upon cAMP-binding suggests isoform-specific differences in cAMP-dependent regulation of PKA activity. (C) 2004 Elsevier Ltd. All rights reserved.