Eichinger L, Noegel A A, Schleicher M
Max-Planck-Institute for Biochemistry, Martinsried, Federal Republic of Germany.
J Cell Biol. 1991 Feb;112(4):665-76. doi: 10.1083/jcb.112.4.665.
Severin from Dictyostelium discoideum is a Ca2(+)-activated actin-binding protein that severs actin filaments, nucleates actin assembly, and caps the fast growing ends of actin filaments. Sequence comparison with functionally related proteins, such as gelsolin, villin, or fragmin revealed highly conserved domains which are thought to be of functional significance. To attribute the different activities of the severin molecule to defined regions, progressively truncated severin polypeptides were constructed. The complete cDNA coding for 362 (DS362) amino acids and five 3' deletions coding for 277 (DS277), 177 (DS177), 151 (DS151), 117 (DS117), or 111 (DS111) amino acids were expressed in Escherichia coli. The proteins were purified to homogeneity and then characterized with respect to their effects on the polymerization or depolymerization kinetics of G- or F-actin solutions and their binding to G-actin. Furthermore, the Ca2+ binding of these proteins was investigated with a 45Ca-overlay assay and by monitoring Ca2(+)-dependent changes in tryptophan fluorescence. Bacterially expressed DS362 showed the same Ca2(+)-dependent activities as native severin. DS277, missing the 85 COOH-terminal amino acids of severin, had lost its strict Ca2+ regulation and displayed a Ca2(+)-independent capping activity, but was still Ca2+ dependent in its severing and nucleating activities. DS151 which corresponded to the first domain of gelsolin or villin had completely lost severing and nucleating properties. However, a residual severing activity of approximately 2% was detectable if 26 amino acids more were present at the COOH-terminal end (DS177). This locates similar to gelsolin the second actin-binding site to the border region between the first and second domain. Measuring the fluorescence enhancement of pyrene-labeled G-actin in the presence of DS111 showed that the first actin-binding site was present in the NH2-terminal 111 amino acids. Extension by six or more amino acids stabilized this actin-binding site in such a way that DS117 and even more pronounced DS151 became Ca2(+)-independent capping proteins. In comparison to many reports on gelsolin we draw the following conclusions. Among the three active actin-binding sites in gelsolin the closely neighboured sites one and two share the F-actin fragmenting function, whereas the actin-binding sites two and three, which are located in far distant domains, collaborate for nucleation. In contrast, severin contains two active actin-binding sites which are next to each other and are responsible for the severing as well as the nucleating function. The single actin-binding site near the NH2-terminus is sufficient for capping of actin filaments.
盘基网柄菌（Dictyostelium discoideum）的肌动蛋白切割蛋白（Severin）是一种Ca2+激活的肌动蛋白结合蛋白，它能切断肌动蛋白丝，引发肌动蛋白组装，并封闭肌动蛋白丝快速生长的末端。与功能相关蛋白（如凝溶胶蛋白、绒毛蛋白或凝溶素）的序列比较显示，存在高度保守的结构域，这些结构域被认为具有功能重要性。为了将肌动蛋白切割蛋白分子的不同活性归因于特定区域，构建了逐步截短的肌动蛋白切割蛋白多肽。编码362个氨基酸（DS362）的完整cDNA以及编码277个（DS277）、177个（DS177）、151个（DS151）、117个（DS117）或111个（DS111）氨基酸的五个3'端缺失cDNA在大肠杆菌中表达。这些蛋白被纯化至同质，然后就其对G-肌动蛋白或F-肌动蛋白溶液聚合或解聚动力学的影响以及它们与G-肌动蛋白的结合进行表征。此外，通过45Ca覆盖分析以及监测色氨酸荧光中Ca2+依赖性变化来研究这些蛋白的Ca2+结合情况。细菌表达的DS362表现出与天然肌动蛋白切割蛋白相同的Ca2+依赖性活性。缺失肌动蛋白切割蛋白85个COOH末端氨基酸的DS277失去了其严格的Ca2+调节，表现出Ca2+非依赖性的封闭活性，但在其切断和成核活性方面仍依赖Ca2+。与凝溶胶蛋白或绒毛蛋白的第一个结构域相对应的DS151完全失去了切断和成核特性。然而，如果COOH末端再多26个氨基酸（DS177），则可检测到约2%的残余切断活性。这将第二个肌动蛋白结合位点类似于凝溶胶蛋白定位到第一和第二结构域之间的边界区域。在DS111存在下测量芘标记的G-肌动蛋白的荧光增强表明，第一个肌动蛋白结合位点存在于NH2末端的111个氨基酸中。六个或更多氨基酸的延伸以这样一种方式稳定了这个肌动蛋白结合位点，使得DS117甚至更明显的DS151成为Ca2+非依赖性的封闭蛋白。与许多关于凝溶胶蛋白的报道相比，我们得出以下结论。在凝溶胶蛋白的三个活性肌动蛋白结合位点中，紧密相邻的位点一和位点二共享F-肌动蛋白片段化功能，而位于遥远结构域中的肌动蛋白结合位点二和位点三协同作用进行成核。相比之下，肌动蛋白切割蛋白包含两个相邻的活性肌动蛋白结合位点，它们负责切断以及成核功能。靠近NH2末端的单个肌动蛋白结合位点足以封闭肌动蛋白丝。
