Yu F X, Zhou D M, Yin H L
Department of Physiology, University of Texas Southwestern Medical Center, Dallas 75235.
J Biol Chem. 1991 Oct 15;266(29):19269-75.
gCap39 is an actin filament end-capping protein which has a threefold repeated domain structure similar to the N-terminal half of gelsolin. However, unlike gelsolin, gCap39 does not sever actin filaments and dissociates completely from filament ends after calcium removal. We have capitalized on these differences to explore the structural basis for actin filament capping, severing, and their regulation. Using truncated gCap39, generated by limited proteolysis or deletion mutagenesis, we found that actin filament capping requires multiple gCap domains, and almost the entire molecule is necessary for optimal activity. gCap39 domain I, like the equivalent domain in gelsolin, contains an actin monomer binding site. gCap39 domains II-III are, however, different from gelsolin in that they do not bind to the side of actin filaments. Since filament side binding is hypothesized to be the first step in severing, lack of side binding may explain why gCap39 does not sever. This is confirmed directly by swapping gCap39 domains II-III for the side-binding gelsolin domains to generate a chimera which severs actin filaments. The chimera is Ca2+ independent in actin filament severing and capping, although gCap39 domain I itself is regulated by Ca2+.
gCap39是一种肌动蛋白丝封端蛋白，其具有与凝溶胶蛋白N端一半相似的三重重复结构域结构。然而，与凝溶胶蛋白不同，gCap39不会切断肌动蛋白丝，并且在去除钙后会从丝末端完全解离。我们利用这些差异来探索肌动蛋白丝封端、切断及其调控的结构基础。使用通过有限蛋白酶解或缺失诱变产生的截短gCap39，我们发现肌动蛋白丝封端需要多个gCap结构域，并且几乎整个分子对于最佳活性都是必需的。gCap39结构域I与凝溶胶蛋白中的等效结构域一样，包含一个肌动蛋白单体结合位点。然而，gCap39结构域II - III与凝溶胶蛋白不同，因为它们不与肌动蛋白丝的侧面结合。由于丝状侧面结合被认为是切断的第一步，缺乏侧面结合可能解释了为什么gCap39不会切断。通过将gCap39结构域II - III替换为侧面结合的凝溶胶蛋白结构域以产生一种切断肌动蛋白丝的嵌合体，这一点得到了直接证实。该嵌合体在肌动蛋白丝切断和封端方面不依赖于Ca2 +，尽管gCap39结构域I本身受Ca2 +调控。
