Identification of a critical ankyrin-binding loop on the cytoplasmic domain of erythrocyte membrane band 3 by crystal structure analysis and site-directed mutagenesis
Identification of a critical ankyrin-binding loop on the cytoplasmic domain of erythrocyte membrane band 3 by crystal structure analysis and site-directed mutagenesis
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DOI：
10.1074/jbc.m211137200
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发表时间：
2003-02-28
影响因子：
4.8
通讯作者：
Low, PS
中科院分区：
文献类型：
--
作者：
Chang, SH;Low, PS
红细胞膜带3（cdb 3）的胞质结构域作为膜组织的中心，与锚蛋白、蛋白4.1、蛋白4.2、血红蛋白、几种糖酵解酶、酪氨酸磷酸酶和酪氨酸激酶p72（syk）等蛋白质相互作用。cdb 3二聚体的晶体学结构显示，残基175-185呈现类似于Na+/K+-ATP酶细胞质表面的推定的锚定结合基序的β-发夹环。为了测试这个发夹环是否构成cdb 3上的锚定结合位点，我们删除了氨基酸175-185，并用Gly-Gly二肽取代了11个残基的环，该二肽桥接了缺失而没有将应变引入结构中。虽然缺失突变体经历了野生型cdb 3所表现出的相同的天然构象变化，并正常地结合其他外周蛋白，但突变体对锚蛋白没有亲和力。这表明暴露的β-发夹转角确实构成了cdb 3上主要的锚定结合位点。其他生物化学研究表明，锚蛋白也停靠在带3的NH 2末端。因此，针对cdb 3的NH 2末端的抗体阻止锚蛋白与cdb 3的结合，而锚蛋白与cdb 3的结合阻止cdb 3在其NH 2末端（主要是Tyr-8）的p72（syk）磷酸化。然而，缺少NH 2-末端50个残基的cdb 3的截短突变体显示与野生型cdb 3相同的结合亲和力。因此，这些数据表明，cdb 3的NH 2末端是近端的，但不是必需的cdb 3-锚蛋白相互作用。
The cytoplasmic domain of erythrocyte membrane band 3 (cdb3) serves as a center of membrane organization, interacting with such proteins as ankyrin, protein 4.1, protein 4.2, hemoglobin, several glycolytic enzymes, a tyrosine phosphatase, and a tyrosine kinase, p72(syk). The crystallographic structure of the cdb3 dimer has revealed that residues 175-185 assume a beta-hairpin loop similar to a putative ankyrin-binding motif at the cytoplasmic surface of the Na+/K+-ATPase. To test whether this hairpin loop constitutes an ankyrin-binding site on cdb3, we have deleted amino acids 175-185 and substituted the 11-residue loop with a Gly-Gly dipeptide that bridges the deletion without introducing strain into the structure. Although the deletion mutant undergoes the same native conformational changes exhibited by wild type cdb3 and binds other peripheral proteins normally, the mutant exhibits no affinity for ankyrin. This suggests that the exposed beta-hairpin turn indeed constitutes a major ankyrin-binding site on cdb3. Other biochemical studies suggest that ankyrin also docks at the NH2 terminus of band 3. Thus, antibodies to the NH2 terminus of cdb3 block ankyrin binding to the cdb3, and ankyrin binding to cdb3 prevents p72(syk) phosphorylation of cdb3 at its NH2 terminus (predominantly at Tyr-8). However, a truncation mutant of cdb3 lacking the NH2-terminal 50 residues displays the same binding affinity as wild type cdb3. These data thus suggest that the NH2 terminus of cdb3 is proximal to but not required for the cdb3-ankyrin interaction.