Missense mutation Lys18Asn in dystrophin that triggers X-linked dilated cardiomyopathy decreases protein stability, increases protein unfolding, and perturbs protein structure, but does not affect protein function.
Missense mutation Lys18Asn in dystrophin that triggers X-linked dilated cardiomyopathy decreases protein stability, increases protein unfolding, and perturbs protein structure, but does not affect protein function.
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DOI：
10.1371/journal.pone.0110439
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发表时间：
2014
期刊：
影响因子：
3.7
通讯作者：
Mallela KM
中科院分区：
文献类型：
--
作者：
Singh SM;Bandi S;Shah DD;Armstrong G;Mallela KM
重要肌肉蛋白抗肌营养不良蛋白的基因突变会引发 X 连锁扩张型心肌病 (XLDCM)。然而，基本蛋白质水平的疾病机制尚不清楚。这些分子知识对于开发 XLDCM 疗法至关重要。我们的主要目标是了解致病突变对肌营养不良蛋白结构和功能的影响。本研究针对的是错义突变 K18N。 K18N 突变发生在 N 端肌动蛋白结合域 (N-ABD)。我们创建并表达了野生型 (WT) N-ABD 及其 K18N 突变体，并纯化至均质。可逆折叠实验表明突变体和WT在重折叠时不会聚集。突变不会影响蛋白质的整体二级结构，蛋白质的圆二色性没有变化表明。然而，突变体在热力学上不如 WT（变性剂熔化）稳定，并且比 WT（停流动力学）展开得更快。尽管具有与 WT 相似的整体二级结构，但与 WT 相比，突变体在许多氨基酸上显示出显着的局部结构变化（异核 NMR 实验）。这些结构变化表明突变的影响在蛋白质结构中长距离传播。与这些结构和稳定性变化相反，突变体对肌动蛋白结合功能没有显着影响，从共沉降和解聚测定中可以明显看出。这些结果总结道，K18N 突变降低了热力学稳定性，加速解折叠，扰乱蛋白质结构，但不影响功能。因此，K18N是稳定性缺陷而不是功能缺陷。稳定性的降低和去折叠的增加会减少可发挥功能的肌营养不良蛋白分子的净数量，这可能会引发 XLDCM。一致的是，XLDCM 患者心肌中肌营养不良蛋白水平降低。
Genetic mutations in a vital muscle protein dystrophin trigger X-linked dilated cardiomyopathy (XLDCM). However, disease mechanisms at the fundamental protein level are not understood. Such molecular knowledge is essential for developing therapies for XLDCM. Our main objective is to understand the effect of disease-causing mutations on the structure and function of dystrophin. This study is on a missense mutation K18N. The K18N mutation occurs in the N-terminal actin binding domain (N-ABD). We created and expressed the wild-type (WT) N-ABD and its K18N mutant, and purified to homogeneity. Reversible folding experiments demonstrated that both mutant and WT did not aggregate upon refolding. Mutation did not affect the protein's overall secondary structure, as indicated by no changes in circular dichroism of the protein. However, the mutant is thermodynamically less stable than the WT (denaturant melts), and unfolds faster than the WT (stopped-flow kinetics). Despite having global secondary structure similar to that of the WT, mutant showed significant local structural changes at many amino acids when compared with the WT (heteronuclear NMR experiments). These structural changes indicate that the effect of mutation is propagated over long distances in the protein structure. Contrary to these structural and stability changes, the mutant had no significant effect on the actin-binding function as evident from co-sedimentation and depolymerization assays. These results summarize that the K18N mutation decreases thermodynamic stability, accelerates unfolding, perturbs protein structure, but does not affect the function. Therefore, K18N is a stability defect rather than a functional defect. Decrease in stability and increase in unfolding decrease the net population of dystrophin molecules available for function, which might trigger XLDCM. Consistently, XLDCM patients have decreased levels of dystrophin in cardiac muscle.
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影响因子：
158.5
作者：
BERKO, BA;SWIFT, M
通讯作者：
SWIFT, M
影响因子：
9.8
作者：
Ferlini, A;Galié, N;Muntoni, F
通讯作者：
Muntoni, F
DOI：
10.1038/77923
发表时间：
2000-08-01
期刊：
NATURE STRUCTURAL BIOLOGY
影响因子：
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作者：
Huang, X;Poy, F;Eck, MJ
通讯作者：
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DOI：
10.1073/pnas.1001517107
发表时间：
2010-05-25
影响因子：
11.1
作者：
Henderson, Davin M.;Lee, Ann;Ervasti, James M.
通讯作者：
Ervasti, James M.
影响因子：
14.8
作者：
Greenfield, Norma J.
通讯作者：
Greenfield, Norma J.