Schlüter K, Schleicher M, Jockusch B M
Cell Biology, Zoological Institute, Technical University of Braunschweig, D-38092 Braunschweig, Germany.
J Cell Sci. 1998 Nov;111 ( Pt 22):3261-73. doi: 10.1242/jcs.111.22.3261.
For a detailed analysis of the profilin-actin interaction, we designed several point mutations in bovine profilin I by computer modeling. The recombinant proteins were analyzed in vitro for their actin-binding properties. Mutant proteins with a putatively higher affinity for actin were produced by attempting to introduce an additional bond to actin. However, these mutants displayed a lower affinity for actin than wild-type profilin, suggesting that additional putative bonds created this way cannot increase profilin's affinity for actin. In contrast, mutants designed to have a reduced affinity for actin by eliminating profilin-actin bonds displayed the desired properties in viscosity assays, while their binding sites for poly(L)proline were still intact. The profilin mutant F59A, with an affinity for actin reduced by one order of magnitude as compared to wild-type profilin, was analyzed further in cells. When microinjected into fibroblasts, F59A colocalized with the endogenous profilin and actin in ruffling areas, suggesting that profilins are targeted to and tethered at these sites by ligands other than actin. Profilin null cells of Dictyostelium were transfected with bovine wild-type profilin I and F59A. Bovine profilin I, although expressed to only approximately 10% of the endogenous profilin level determined for wild-type Dictyostelium, caused a substantial rescue of the defects observed in profilin null amoebae, as seen by measuring the growth of colony surface areas and the percentage of polynucleated cells. The mutant protein was much less effective. These results emphasize the highly conserved biological function of profilins with low sequence homology, and correlate specifically their actin-binding capacity with cell motility and proliferation.
为了详细分析肌动蛋白结合蛋白与肌动蛋白的相互作用，我们通过计算机建模在牛肌动蛋白结合蛋白I中设计了几个点突变。对重组蛋白的肌动蛋白结合特性进行了体外分析。通过尝试引入与肌动蛋白的额外键来产生对肌动蛋白具有假定更高亲和力的突变蛋白。然而，这些突变体对肌动蛋白的亲和力低于野生型肌动蛋白结合蛋白，这表明以这种方式产生的额外假定键并不能增加肌动蛋白结合蛋白对肌动蛋白的亲和力。相反，通过消除肌动蛋白结合蛋白 - 肌动蛋白键而设计成对肌动蛋白亲和力降低的突变体在粘度测定中表现出所需的特性，而它们对聚（L）脯氨酸的结合位点仍然完整。与野生型肌动蛋白结合蛋白相比，肌动蛋白结合蛋白突变体F59A对肌动蛋白的亲和力降低了一个数量级，在细胞中对其进行了进一步分析。当微注射到成纤维细胞中时，F59A在内源性肌动蛋白结合蛋白和肌动蛋白的褶皱区域共定位，这表明肌动蛋白结合蛋白通过肌动蛋白以外的配体靶向并拴系在这些位点。用牛野生型肌动蛋白结合蛋白I和F59A转染盘基网柄菌的肌动蛋白结合蛋白缺失细胞。通过测量菌落表面积的生长和多核细胞的百分比可以看出，牛肌动蛋白结合蛋白I虽然表达水平仅为野生型盘基网柄菌中内源性肌动蛋白结合蛋白水平的约10％，但却能显著挽救在肌动蛋白结合蛋白缺失变形虫中观察到的缺陷。突变蛋白的效果要差得多。这些结果强调了肌动蛋白结合蛋白具有高度保守的生物学功能，其序列同源性较低，并将它们的肌动蛋白结合能力与细胞运动和增殖具体关联起来。
