Distinct interaction modes of an AKAP bound to two regulatory subunit isoforms of protein kinase A revealed by amide hydrogen/deuterium exchange
Distinct interaction modes of an AKAP bound to two regulatory subunit isoforms of protein kinase A revealed by amide hydrogen/deuterium exchange
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DOI：
10.1110/ps.051687305
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发表时间：
2005-12-01
期刊：
影响因子：
8
通讯作者：
Woods, VL
中科院分区：
文献类型：
--
作者：
Burns-Hamuro, LL;Hamuro, Y;Woods, VL
与蛋白激酶A (PKA) II型（RII α）异构体对接的AKAP的结构已经被很好地表征，但目前还没有与I型（RI α）异构体对接的AKAP的详细结构信息。双特异性AKAP2 （D-AKAP2）在纳摩尔范围内与这两种异构体结合，这为我们提供了一个利用共同的停靠配体来表征AKAP结合的异构体选择性的机会。采用氢/氘（H/D）交换结合质谱（DXMS）技术研究了连接到RI α和RII α D/D结构域的D- akap2 α -螺旋a激酶结合（AKB）基序的骨干结构变化。确定了络合物形成时的保护区域和H/D交换对每个络合物相互作用伙伴的保护幅度。与RII α相比，AKB配体的骨干在与RI α结合时受到更多的保护，这表明对接的AKB配体具有更高的螺旋稳定性。这与AKAP在RI α对接表面诱导的更广泛的骨干保护区域相结合，表明AKB配体与RI α结合时具有更多的结合约束。这与RII α形成对比，后者具有预先形成的局部结合表面。这些不同的AKAP结合模式可能有助于RI α AKAP对接表面更具歧视性。在缺乏高分辨率结构的情况下，DXMS为理解结合特异性提供了有价值的结构信息，并且可以很容易地应用于其他蛋白质-配体和蛋白质-蛋白质相互作用。
The structure of an AKAP docked to the dimerization/docking (D/D) domain of the type II (RII alpha) isoform of protein kinase A (PKA) has been well characterized, but there currently is no detailed structural information of an AKAP docked to the type I (RI alpha) isoform. Dual-specific AKAP2 (D-AKAP2) binds in the nanomolar range to both isoforms and provided us with an opportunity to characterize the isoform-selective nature of AKAP binding using a common docked ligand. Hydrogen/ deuterium (H/D) exchange combined with mass spectrometry (DXMS) was used to probe backbone structural changes of an alpha-helical A-kinase binding (AKB) motif from D-AKAP2 docked to both RI alpha and RII alpha D/D domains. The region of protection upon complex formation and the magnitude of protection from H/D exchange were determined for both interacting partners in each complex. The backbone of the AKB ligand was more protected when bound to RI alpha compared to RII alpha, suggesting an increased helical stabilization of the docked AKB ligand. This combined with a broader region of backbone protection induced by the AKAP on the docking surface of RI alpha indicated that there were more binding constraints for the AKB ligand when bound to RI alpha. This was in contrast to RII alpha, which has a preformed, localized binding surface. These distinct modes of AKAP binding may contribute to the more discriminating nature of the RI alpha AKAP-docking surface. DXMS provides valuable structural information for understanding binding specificity in the absence of a high-resolution structure, and can readily be applied to other protein-ligand and protein-protein interactions.