Walder J A, Chatterjee R, Steck T L, Low P S, Musso G F, Kaiser E T, Rogers P H, Arnone A
J Biol Chem. 1984 Aug 25;259(16):10238-46.
Previous studies point to the acidic amino-terminal segment of band 3, the anion transport protein of the red cell, as the common binding site for hemoglobin and several of the glycolytic enzymes to the erythrocyte membrane. We now report on the interaction of hemoglobin with the synthetic peptide AcM-E-E-L-Q-D-D-Y-E-D-E, corresponding to the first 11 residues of band 3, and with the entire 43,000-Da cytoplasmic domain of the protein. In the presence of increasing concentrations of the peptide, the oxygen binding curve for hemoglobin is shifted progressively to the right, indicating that the peptide binds preferentially to deoxyhemoglobin. The dissociation constant for the deoxyhemoglobin-peptide complex at pH 7.2 in the presence of 100 mM NaCl is 0.31 mM. X-ray crystallographic studies were carried out to determine the exact mode of binding of the peptide to deoxyhemoglobin. The difference electron density map of the deoxyhemoglobin-peptide complex at 5 A resolution showed that the binding site extends deep (approximately 18 A) into the central cavity between the beta chains, along the dyad symmetry axis, and includes Arg 104 beta 1 and Arg 104 beta 2 as well as most of the basic residues within the 2,3-diphosphoglycerate binding site. The peptide appears to have an extended conformation with only 5 to 7 of the 11 residues in contact with hemoglobin. In agreement with the crystallographic studies, binding of the peptide to deoxyhemoglobin was blocked by cross-linking the beta chains at the entrance to the central cavity. Oxygen equilibrium studies showed that the isolated cytoplasmic fragment of band 3 also binds preferentially to deoxyhemoglobin. The binding of the 43,000-Da fragment to hemoglobin was inhibited in the cross-linked derivative indicating that the acidic amino-terminal residues in the intact cytoplasmic domain also bind within the central cavity of the hemoglobin tetramer.
先前的研究指出，红细胞阴离子转运蛋白带3的酸性氨基末端片段是血红蛋白和几种糖酵解酶与红细胞膜的共同结合位点。我们现在报告血红蛋白与对应于带3前11个残基的合成肽AcM-E-E-L-Q-D-D-Y-E-D-E以及该蛋白完整的43000道尔顿细胞质结构域之间的相互作用。在肽浓度不断增加的情况下，血红蛋白的氧结合曲线逐渐向右移动，表明该肽优先与脱氧血红蛋白结合。在100 mM NaCl存在下，pH 7.2时脱氧血红蛋白 - 肽复合物的解离常数为0.31 mM。进行了X射线晶体学研究以确定肽与脱氧血红蛋白的精确结合模式。5埃分辨率下脱氧血红蛋白 - 肽复合物的差分电子密度图显示，结合位点沿着二重对称轴深入到β链之间的中心腔深处（约18埃），包括Arg 104β1和Arg 104β2以及2,3 - 二磷酸甘油酸结合位点内的大部分碱性残基。该肽似乎具有伸展构象，11个残基中只有5至7个与血红蛋白接触。与晶体学研究一致，通过在中心腔入口处交联β链，肽与脱氧血红蛋白的结合被阻断。氧平衡研究表明，分离的带3细胞质片段也优先与脱氧血红蛋白结合。在交联衍生物中，43000道尔顿片段与血红蛋白的结合受到抑制，这表明完整细胞质结构域中的酸性氨基末端残基也在血红蛋白四聚体的中心腔内结合。
