Amino-terminal basic residues of Src mediate membrane binding through electrostatic interaction with acidic phospholipids.

Membrane targeting of pp60src (Src) is mediated by its myristoylated amino terminus. We demonstrate that, in addition to myristate, six basic residues in the amino terminus are essential for high-affinity binding to the lipid bilayer via electrostatic interaction with acidic phospholipids. Specifically, c-Src was shown to bind 2500-fold more strongly to vesicles composed of the physiological ratio of 2:1 phosphatidylcholine (PC)/phosphatidylserine (PS) than to neutral PC bilayer vesicles. The apparent Kd for binding of c-Src to the PC/PS bilayer was 6 x 10(-7) M. This interaction is sufficiently strong to account for c-Src membrane targeting. Mutants of c-Src in which the amino-terminal basic residues were replaced by neutral asparagine residues exhibited binding isotherms approaching that of wild-type binding to neutral bilayers (apparent Kd of 2 x 10(-3) M). The transforming v-Src and activated c-Src (Y527F) proteins also bound more strongly to PC/PS bilayers (apparent Kd of approximately 1 x 10(-5) M) than to neutral PC bilayers. In vivo experiments with Src mutants confirmed the role of positive charge in mediating membrane binding and cellular transformation. IMAGES:

pp60src（Src）的膜靶向作用由其肉豆蔻酰化的氨基末端介导。本研究证实，除肉豆蔻酸修饰外，氨基末端的6个碱性残基对于通过与酸性磷脂的静电相互作用实现与脂质双分子层的高亲和力结合至关重要。具体而言，实验显示c-Src与生理比例为2:1的磷脂酰胆碱（PC）/磷脂酰丝氨酸（PS）组成的囊泡的结合强度，是其与中性PC脂质双分子层囊泡结合强度的2500倍。c-Src与PC/PS双分子层结合的表观解离常数（Kd）为6×10^-7 M。该相互作用的强度足以解释c-Src的膜靶向机制。将氨基末端碱性残基替换为中性天冬酰胺残基的c-Src突变体，其结合等温曲线接近野生型c-Src与中性双分子层的结合曲线（表观解离常数为2×10^-3 M）。致癌性v-Src与激活型c-Src（Y527F）蛋白同样对PC/PS双分子层表现出更强的结合能力（表观解离常数约为1×10^-5 M），其结合强度高于与中性PC双分子层的结合。针对Src突变体的体内实验证实，正电荷在介导膜结合与细胞转化过程中发挥了关键作用。图像：