Expression and Purification of ZASP Subdomains and Clinically Important Isoforms: High-Affinity Binding to G-Actin.
Expression and Purification of ZASP Subdomains and Clinically Important Isoforms: High-Affinity Binding to G-Actin.
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DOI：
10.1021/acs.biochem.7b00067
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发表时间：
2017-04-11
期刊：
影响因子：
2.9
通讯作者：
Wingfield PT
中科院分区：
文献类型：
--
作者：
Watts NR;Zhuang X;Kaufman JD;Palmer IW;Dearborn AD;Coscia S;Blech-Hermoni Y;Alfano C;Pastore A;Mankodi A;Wingfield PT
Z盘相关的选择性剪接的含PDZ基序的蛋白（ZASP）是肌节的主要成分。骨骼肌中ZASP的三种常见亚型是通过外显子9和10的选择性剪接产生的。长同种型，具有（ZASP-L）或缺乏外显子10（ZASP-LΔ ex 10），包括N-末端PDZ结构域，具有保守基序（ZM）的肌动蛋白结合区（ABR）和三个C-末端LIM结构域。短同种型（ZASP-S）缺乏LIM结构域。ZASP-LΔ ex 10的ZM内的突变A147 T和A165 V引起肌原纤维肌病，但其机制尚不清楚。我们已经准备好这些蛋白质，它们的ABR，和各自的突变体的重组形式，其特征在于它们的bioprophilically，并通过表面等离子体共振和电子显微镜分析其肌动蛋白结合特性。所有的蛋白质都是物理上均匀的单体，并具有与部分折叠构象一致的圆二色性光谱。ZASP-S和PDZ结构域的NMR HSQC谱的比较表明，ABR是非结构化的。ZASP-S及其突变体和ZASP-LΔ ex 10都以高亲和力（Kd = 10−8至10−9 M）与固定化的G-肌动蛋白结合。缺失外显子10的肌动蛋白结合区（ABRΔ10）的构建体结合亲和力较低（Kd = 10−7 M），但保留外显子10的构建体（ABR+10）结合亲和力较弱（Kd = 10−5 M）。ZASP-S和ABRΔ10也诱导F-肌动蛋白和阵列形成，即使在低离子强度和不存在KCl和Mg 2+离子的条件下。有趣的是，ZM突变A147 T和A165 V不影响上述任何结果。
Z-disc-associated, alternatively spliced, PDZ motif-containing protein (ZASP) is a principal component of the sarcomere. The three prevalent isoforms of ZASP in skeletal muscle are generated by alternative splicing of exons 9 and 10. The long isoforms, either having (ZASP-L) or lacking exon 10 (ZASP-LΔex10), include an N-terminal PDZ domain, an actin-binding region (ABR) with a conserved motif (ZM), and three C-terminal LIM domains. The short isoform (ZASP-S) lacks the LIM domains. Mutations, A147T and A165V, within the ZM of ZASP-LΔex10 cause myofibrillar myopathy, but the mechanism is unknown. We have prepared these proteins, their ABR, and the respective mutant variants in recombinant form, characterized them biophysically, and analyzed their actin-binding properties by surface plasmon resonance and electron microscopy. All the proteins were physically homogeneous and monomeric and had circular dichroic spectra consistent with partially folded conformations. Comparison of the NMR HSQC spectra of ZASP-S and the PDZ domain showed that the ABR is unstructured. ZASP-S and its mutant variants and ZASP-LΔex10 all bound to immobilized G-actin with high affinity (Kd ≈ 10−8 to 10−9 M). Constructs of the isolated actin-binding region missing exon 10 (ABRΔ10) bound with lower affinity (Kd ≈ 10−7 M), but those retaining exon 10 (ABR+10) did so only weakly (Kd ≈ 10−5 M). ZASP-S, and the ABRΔ10, also induced F-actin and array formation, even in conditions of low ionic strength and in the absence of KCl and Mg2+ ions. Interestingly, the ZM mutations A147T and A165V did not affect any of the results described above.
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