Schwebach Christopher L, Agrawal Richa, Lindert Steffen, Kudryashova Elena, Kudryashov Dmitri S
Department of Chemistry and Biochemistry, The Ohio State University, Columbus, OH 43210, USA; Molecular, Cellular, and Developmental Biology Program, The Ohio State University, Columbus, OH 43210, USA.
Department of Chemistry and Biochemistry, The Ohio State University, Columbus, OH 43210, USA.
J Mol Biol. 2017 Aug 4;429(16):2490-2508. doi: 10.1016/j.jmb.2017.06.021. Epub 2017 Jul 8.
The actin cytoskeleton is a complex network controlled by a vast array of intricately regulated actin-binding proteins. Human plastins (PLS1, PLS2, and PLS3) are evolutionary conserved proteins that non-covalently crosslink actin filaments into tight bundles. Through stabilization of such bundles, plastins contribute, in an isoform-specific manner, to the formation of kidney and intestinal microvilli, inner ear stereocilia, immune synapses, endocytic patches, adhesion contacts, and invadosomes of immune and cancer cells. All plastins comprise an N-terminal Ca-binding regulatory headpiece domain followed by two actin-binding domains (ABD1 and ABD2). Actin bundling occurs due to simultaneous binding of both ABDs to separate actin filaments. Bundling is negatively regulated by Ca, but the mechanism of this inhibition remains unknown. In this study, we found that the bundling abilities of PLS1 and PLS2 were similarly sensitive to Ca (pCa ~6.4), whereas PLS3 was less sensitive (pCa ~5.9). At the same time, all three isoforms bound to F-actin in a Ca-independent manner, suggesting that binding of only one of the ABDs is inhibited by Ca. Using limited proteolysis and mass spectrometry, we found that in the presence of Ca the EF-hands of human plastins bound to an immediately adjacent sequence homologous to canonical calmodulin-binding peptides. Furthermore, our data from differential centrifugation, Förster resonance energy transfer, native electrophoresis, and chemical crosslinking suggest that Ca does not affect ABD1 but inhibits the ability of ABD2 to interact with actin. A structural mechanism of signal transmission from Ca to ABD2 through EF-hands remains to be established.
肌动蛋白细胞骨架是一个复杂的网络，受大量复杂调控的肌动蛋白结合蛋白控制。人类丝束蛋白（PLS1、PLS2和PLS3）是进化保守的蛋白，它们将肌动蛋白丝非共价交联成紧密的束状结构。通过稳定这些束状结构，丝束蛋白以异构体特异性的方式，促进肾和肠微绒毛、内耳静纤毛、免疫突触、内吞小斑、黏附连接以及免疫细胞和癌细胞的侵袭性伪足的形成。所有丝束蛋白都包含一个N端钙结合调节头部结构域，其后跟着两个肌动蛋白结合结构域（ABD1和ABD2）。肌动蛋白束的形成是由于两个ABD同时与分开的肌动蛋白丝结合。束的形成受钙的负调控，但其抑制机制仍不清楚。在本研究中，我们发现PLS1和PLS2的束形成能力对钙同样敏感（pCa约为6.4），而PLS3较不敏感（pCa约为5.9）。同时，所有三种异构体都以钙不依赖的方式与F-肌动蛋白结合，这表明只有一个ABD的结合受到钙的抑制。使用有限蛋白酶解和质谱分析，我们发现在有钙存在的情况下，人类丝束蛋白的EF手结构域与一个紧邻的、与典型钙调蛋白结合肽同源的序列结合。此外，我们从差速离心、Förster共振能量转移、非变性电泳和化学交联得到的数据表明，钙不影响ABD1，但抑制ABD2与肌动蛋白相互作用的能力。从钙通过EF手结构域向ABD2传递信号的结构机制仍有待确定。
