Cyclic AMP analog blocks kinase activation by stabilizing inactive conformation: conformational selection highlights a new concept in allosteric inhibitor design.
Cyclic AMP analog blocks kinase activation by stabilizing inactive conformation: conformational selection highlights a new concept in allosteric inhibitor design.
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DOI：
10.1074/mcp.m110.004390
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发表时间：
2011-03-01
期刊：
影响因子：
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通讯作者：
Anand, Ganesh S
中科院分区：
文献类型：
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作者：
Badireddy, Suguna;Yunfeng, Gao;Anand, Ganesh S
蛋白激酶A的调节（R）亚基用于以cAMP依赖性方式调节蛋白激酶A的活性，并且以两种不同且结构上不同的端点cAMP结合的“B”和C亚基结合的“H”构象存在。在这里，我们报告的cAMP行动的机制细节，尚未通过一个独特的方法结合X-射线晶体学与结构蛋白质组学方法，酰胺氢/氘交换和离子迁移率质谱，适用于立体特异性cAMP硫代磷酸酯类似物和拮抗剂（（Rp）-cAMP）的研究。X射线晶体学显示cAMP结合的R亚基呈B形式，但令人惊讶的是，拮抗剂Rp-cAMPS结合的R亚基以H构象结晶，此前认为这仅由C亚基结合诱导。载脂蛋白R-亚基也以B形式结晶，但酰胺交换质谱显示载脂蛋白、激动剂和拮抗剂R-亚基结合状态之间存在较大差异。进一步的离子迁移率揭示了载脂蛋白R-亚基作为多种构象的集合，其碰撞横截面积跨越激动剂和拮抗剂结合状态。因此，与早期的研究，解释了cAMP的行动，通过“诱导适合”单独的基础上，我们报告的证据构象选择，其中的配体无载脂蛋白形式的R-亚基存在作为一个整体的B和H构象。尽管cAMP优先结合B构象，但Rp-cAMPS有趣地结合H构象。这揭示了环状磷酸酯的赤道氧在介导从H到B形式的构象转变中的独特重要性，突出了基于合理结构的药物设计的新方法。理想的抑制剂如Rp-cAMPS是通过单独满足所有“结合”约束而不诱导活化所需的构象变化来优先“选择”靶蛋白的无活性构象的那些。
The regulatory (R) subunit of protein kinase A serves to modulate the activity of protein kinase A in a cAMP-dependent manner and exists in two distinct and structurally dissimilar, end point cAMP-bound "B" and C-subunit-bound "H"-conformations. Here we report mechanistic details of cAMP action as yet unknown through a unique approach combining x-ray crystallography with structural proteomics approaches, amide hydrogen/deuterium exchange and ion mobility mass spectrometry, applied to the study of a stereospecific cAMP phosphorothioate analog and antagonist((Rp)-cAMPS). X-ray crystallography shows cAMP-bound R-subunit in the B form but surprisingly the antagonist Rp-cAMPS-bound R-subunit crystallized in the H conformation, which was previously assumed to be induced only by C-subunit-binding. Apo R-subunit crystallized in the B form as well but amide exchange mass spectrometry showed large differences between apo, agonist and antagonist-bound states of the R-subunit. Further ion mobility reveals the apo R-subunit as an ensemble of multiple conformations with collisional cross-sectional areas spanning both the agonist and antagonist-bound states. Thus contrary to earlier studies that explained the basis for cAMP action through "induced fit" alone, we report evidence for conformational selection, where the ligand-free apo form of the R-subunit exists as an ensemble of both B and H conformations. Although cAMP preferentially binds the B conformation, Rp-cAMPS interestingly binds the H conformation. This reveals the unique importance of the equatorial oxygen of the cyclic phosphate in mediating conformational transitions from H to B forms highlighting a novel approach for rational structure-based drug design. Ideal inhibitors such as Rp-cAMPS are those that preferentially "select" inactive conformations of target proteins by satisfying all "binding" constraints alone without inducing conformational changes necessary for activation.