Interdomain Linker Determines Primarily the Structural Stability of Dystrophin and Utrophin Tandem Calponin-Homology Domains Rather than Their Actin-Binding Affinity
Interdomain Linker Determines Primarily the Structural Stability of Dystrophin and Utrophin Tandem Calponin-Homology Domains Rather than Their Actin-Binding Affinity
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DOI：
10.1021/acs.biochem.5b00741
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发表时间：
2015-09-08
期刊：
影响因子：
2.9
通讯作者：
Mallela, Krishna M. G.
中科院分区：
文献类型：
--
作者：
Bandi, Swati;Singh, Surinder M.;Mallela, Krishna M. G.
串联型钙调蛋白同源结构域是蛋白质中最常见的肌动蛋白结合结构域。然而，其功能背后的结构原理知之甚少。这些串联结构域以多种构象存在，具有不同程度的CH结构域间相互作用。Dystrophin和utrophin串联CH结构域具有较高的序列相似性（相似性为82%），但在结构稳定性和肌动蛋白结合亲和力方面存在差异。我们研究了两个串联CH结构域之间的构象差异是否可以解释它们的稳定性和肌动蛋白结合的差异。Dystrophin的串联CH结构域比utrophin的串联CH结构域稳定约4kcal/mol。肌营养不良蛋白和肌营养不良蛋白的单个CH结构域具有相同的结构，但在结构域间接头周围的相对方向上不同。我们交换了肌营养不良蛋白和肌营养不良蛋白串联CH结构域之间的接头。具有utrophin接头的Dystrophin串联CH结构域（DUL）具有与utrophin串联CH结构域类似的稳定性。具有抗肌萎缩蛋白连接子（UDL）的Utrophin串联CH结构域具有与抗肌萎缩蛋白串联CH结构域相似的稳定性。Dystrophin串联CH结构域与F-肌动蛋白的结合比utrophin弱30倍。接头交换后，DUL的结合亲和力是抗肌萎缩蛋白串联CH结构域的两倍。类似地，UDL的结合亲和力是utrophin串联CH结构域的一半。然而，由于接头交换的结合自由能的变化比相应的解折叠自由能的变化低一个数量级。这些结果表明，接头区主要决定串联CH结构域的结构稳定性，而不是他们的肌动蛋白结合亲和力。
Tandem calponin-homology (CH) domains are the most common actin-binding domains in proteins. However, structural principles underlying their function are poorly understood. These tandem domains exist in multiple conformations with varying degrees of inter-CH-domain interactions. Dystrophin and utrophin tandem CH domains share high sequence similarity (similar to 82%), yet differ in their structural stability and actin-binding affinity. We examined whether the conformational differences between the two tandem CH domains can explain differences in their stability and actin binding. Dystrophin tandem CH domain is more stable by similar to 4 kcal/mol than that of utrophin. Individual CH domains of dystrophin and utrophin have identical structures but differ in their relative orientation around the interdomain linker. We swapped the linkers between dystrophin and utrophin tandem CH domains. Dystrophin tandem CH domain with utrophin linker (DUL) has similar stability as that of utrophin tandem CH domain. Utrophin tandem CH domain with dystrophin linker (UDL) has similar stability as that of dystrophin tandem CH domain. Dystrophin tandem CH domain binds to F-actin similar to 30 times weaker than that of utrophin. After linker swapping, DUL has twice the binding affinity as that of dystrophin tandem CH domain. Similarly, UDL has half the binding affinity as that of utrophin tandem CH domain. However, changes in binding free energies due to linker swapping are much lower by an order of magnitude compared to the corresponding changes in unfolding free energies. These results indicate that the linker region determines primarily the structural stability of tandem CH domains rather than their actin-binding affinity.