Stolt Peggy C, Vardar Didem, Blacklow Stephen C
Department of Pathology, Harvard Medical School and Brigham and Women's Hospital, 77 Avenue Louis Pasteur, Boston, Massachusetts 02115, USA.
Biochemistry. 2004 Aug 31;43(34):10979-87. doi: 10.1021/bi049092l.
While typical intracellular protein modules have only one ligand-binding site, there are rare examples of single modules that bind two different ligands at distinct binding sites. Here we present a detailed mutational and energetic analysis of one such domain, the phosphotyrosine binding (PTB) domain of Disabled-1 (Dab1), which binds to both peptide and phosphoinositide (PI) ligands simultaneously at structurally distinct binding sites. Through the techniques of isothermal titration calorimetry (ITC), analysis of Dab1 PTB domain mutants, and nuclear magnetic resonance (NMR), we have evaluated the characteristics of binding of the Dab1 PTB domain to various peptide and PI ligands. These studies reveal that the presence of saturating concentrations of one ligand has little effect on the binding constant for a second ligand at the other site. In addition, proteins with single-point mutations in the peptide-binding site retain native affinity for PI ligands, while proteins with mutations that prevent PI binding retain native affinity for peptide. NMR titrations show that the final structure of the ternary complex is the same independent of the order of addition of the two ligands. Together, these studies show that binding of peptide and PI ligands is energetically independent and noncooperative.
虽然典型的细胞内蛋白质模块只有一个配体结合位点，但也有罕见的单个模块在不同结合位点结合两种不同配体的例子。在这里，我们对一个这样的结构域进行了详细的突变和能量分析，即失活蛋白1（Dab1）的磷酸酪氨酸结合（PTB）结构域，它在结构上不同的结合位点同时结合肽和磷酸肌醇（PI）配体。通过等温滴定量热法（ITC）、Dab1 PTB结构域突变体分析以及核磁共振（NMR）技术，我们评估了Dab1 PTB结构域与各种肽和PI配体的结合特性。这些研究表明，一种配体的饱和浓度的存在对另一个位点上第二种配体的结合常数影响很小。此外，在肽结合位点有单点突变的蛋白质对PI配体保留天然亲和力，而具有阻止PI结合的突变的蛋白质对肽保留天然亲和力。NMR滴定表明，三元复合物的最终结构与两种配体添加顺序无关。总之，这些研究表明肽和PI配体的结合在能量上是独立且非协同的。
