The auto-inhibitory role of the EPAC hinge helix as mapped by NMR.
The auto-inhibitory role of the EPAC hinge helix as mapped by NMR.
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DOI：
10.1371/journal.pone.0048707
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发表时间：
2012
期刊：
影响因子：
3.7
通讯作者：
Melacini G
中科院分区：
文献类型：
--
作者：
Selvaratnam R;Mazhab-Jafari MT;Das R;Melacini G
环amp结合域（CBD）是cAMP （EPAC）激活的交换蛋白的中心调控单元。在没有变构效应物cAMP的情况下，CBD维持EPAC处于自抑制状态。当cAMP与CBD结合时，这种自我抑制被释放，导致EPAC激活。研究表明，这种camp依赖性激活过程的一个关键特征是CBD c端结构保守的铰链螺旋的部分不稳定。然而，这种螺旋结构在自抑制中的作用目前尚未完全了解。在这里，我们利用一系列模仿cAMP引起的铰链螺旋不稳定的渐进缺失突变来表明这种螺旋也是apo-EPAC的关键自抑制元件。利用最近开发的核磁共振化学位移投影和协方差分析方法评估了缺失突变对自抑制载脂蛋白/非活性和载脂蛋白/活性平衡的影响。我们的研究结果表明，即使在没有cAMP的情况下，铰链螺旋的c端区域与CBD的其他保守变构结构元件紧密耦合，并且破坏铰链螺旋稳定性的扰动使自抑制平衡向载脂蛋白/活性构象转移。这些发现解释了明显违反直觉的观察结果，即cAMP与较短的EPAC结构比较长的EPAC结构结合更紧密。这些结果与一般的cbd相关，并解释了为什么底物使含cbd的系统对cAMP敏感。此外，这里提出的核磁共振分析有望在定量评估突变如何影响构象平衡方面普遍有用。
The cyclic-AMP binding domain (CBD) is the central regulatory unit of exchange proteins activated by cAMP (EPAC). The CBD maintains EPAC in a state of auto-inhibition in the absence of the allosteric effector, cAMP. When cAMP binds to the CBD such auto-inhibition is released, leading to EPAC activation. It has been shown that a key feature of such cAMP-dependent activation process is the partial destabilization of a structurally conserved hinge helix at the C-terminus of the CBD. However, the role of this helix in auto-inhibition is currently not fully understood. Here we utilize a series of progressive deletion mutants that mimic the hinge helix destabilization caused by cAMP to show that such helix is also a pivotal auto-inhibitory element of apo-EPAC. The effect of the deletion mutations on the auto-inhibitory apo/inactive vs. apo/active equilibrium was evaluated using recently developed NMR chemical shift projection and covariance analysis methods. Our results show that, even in the absence of cAMP, the C-terminal region of the hinge helix is tightly coupled to other conserved allosteric structural elements of the CBD and perturbations that destabilize the hinge helix shift the auto-inhibitory equilibrium toward the apo/active conformations. These findings explain the apparently counterintuitive observation that cAMP binds more tightly to shorter than longer EPAC constructs. These results are relevant for CBDs in general and rationalize why substrates sensitize CBD-containing systems to cAMP. Furthermore, the NMR analyses presented here are expected to be generally useful to quantitatively evaluate how mutations affect conformational equilibria.
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