Watts Norman R, Zhuang Xiaolei, Kaufman Joshua D, Palmer Ira W, Dearborn Altaira D, Coscia Stephen, Blech-Hermoni Yotam, Alfano Caterina, Pastore Annalisa, Mankodi Ami, Wingfield Paul T
Department of Clinical and Basic Neuroscience, King's College London , London SE5 8AF, United Kingdom.
Biochemistry. 2017 Apr 11;56(14):2061-2070. doi: 10.1021/acs.biochem.7b00067. Epub 2017 Apr 3.
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 (K ≈ 10 to 10 M). Constructs of the isolated actin-binding region missing exon 10 (ABRΔ10) bound with lower affinity (K ≈ 10 M), but those retaining exon 10 (ABR+10) did so only weakly (K ≈ 10 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 Mg ions. Interestingly, the ZM mutations A147T and A165V did not affect any of the results described above.
Z盘相关的、可变剪接的、含PDZ基序的蛋白（ZASP）是肌节的主要成分。骨骼肌中ZASP的三种常见异构体是由外显子9和10的可变剪接产生的。长异构体，要么含有（ZASP-L）要么缺少外显子10（ZASP-LΔex10），包括一个N端PDZ结构域、一个具有保守基序（ZM）的肌动蛋白结合区域（ABR）和三个C端LIM结构域。短异构体（ZASP-S）缺少LIM结构域。ZASP-LΔex10的ZM内的突变A147T和A165V会导致肌原纤维肌病，但其机制尚不清楚。我们已经以重组形式制备了这些蛋白、它们的ABR以及各自的突变变体，对它们进行了生物物理表征，并通过表面等离子体共振和电子显微镜分析了它们的肌动蛋白结合特性。所有蛋白在物理上都是均一的且为单体，并且具有与部分折叠构象一致的圆二色光谱。ZASP-S和PDZ结构域的核磁共振HSQC谱比较表明ABR是无结构的。ZASP-S及其突变变体以及ZASP-LΔex10都以高亲和力（K≈(10^{-6})至(10^{-7})M）结合到固定化的G-肌动蛋白上。缺失外显子10的分离肌动蛋白结合区域（ABRΔ10）的构建体以较低亲和力（K≈(10^{-5})M）结合，但保留外显子10的构建体（ABR + 10）结合较弱（K≈(10^{-4})M）。即使在低离子强度以及没有KCl和Mg离子的条件下，ZASP-S和ABRΔ10也能诱导F-肌动蛋白和成束形成。有趣的是，ZM突变A147T和A165V并不影响上述任何结果。
