Li S C, Songyang Z, Vincent S J, Zwahlen C, Wiley S, Cantley L, Kay L E, Forman-Kay J, Pawson T
Program in Molecular Biology and Cancer, Samuel Lunenfeld Research Institute, Mount Sinai Hospital, Toronto, Ontario M5G 1X5, Canada.
Proc Natl Acad Sci U S A. 1997 Jul 8;94(14):7204-9. doi: 10.1073/pnas.94.14.7204.
The phosphotyrosine-binding (PTB) domain is a recently identified protein module that has been characterized as binding to phosphopeptides containing an NPXpY motif (X = any amino acid). We describe here a novel peptide sequence recognized by the PTB domain from Drosophila Numb (dNumb), a protein involved in cell fate determination and asymmetric cell division during the development of the Drosophila nervous system. Using a Tyr-oriented peptide library to screen for ligands, the dNumb PTB domain was found to bind selectively to peptides containing a YIGPYphi motif (phi represents a hydrophobic residue). A synthetic peptide containing this sequence bound specifically to the isolated dNumb PTB domain in solution with a dissociation constant (Kd) of 5.78 +/- 0.74 microM. Interestingly, the affinity of this peptide for the dNumb PTB domain was increased (Kd = 1.41 +/- 0.10 microM) when the second tyrosine in the sequence was phosphorylated. Amino acid substitution studies of the phosphopeptide demonstrated that a core motif of sequence GP(p)Y is required for high-affinity binding to the dNumb PTB domain. Nuclear magnetic resonance experiments performed on isotopically labeled protein complexed with either Tyr- or pTyr-containing peptides suggest that the same set of amino acids in the dNumb PTB domain is involved in binding both phosphorylated and nonphosphorylated forms of the peptide. The in vitro selectivity of the dNumb PTB domain is therefore markedly different from those of the Shc and IRS-1 PTB domains, in that it interacts preferentially with a GP(p)Y motif, rather than NPXpY, and does not absolutely require ligand phosphorylation for binding. Our results suggest that the PTB domain is a versatile protein module, capable of exhibiting varied binding specificities.
磷酸酪氨酸结合（PTB）结构域是最近发现的一种蛋白质模块，其特征是能与含有NPXpY基序（X = 任意氨基酸）的磷酸肽结合。我们在此描述一种由果蝇Numb（dNumb）的PTB结构域识别的新型肽序列，dNumb是一种在果蝇神经系统发育过程中参与细胞命运决定和不对称细胞分裂的蛋白质。通过使用以酪氨酸为导向的肽库筛选配体，发现dNumb PTB结构域选择性地结合含有YIGPYphi基序（phi代表疏水残基）的肽。一种含有该序列的合成肽在溶液中与分离的dNumb PTB结构域特异性结合，解离常数（Kd）为5.78 +/- 0.74微摩尔。有趣的是，当该序列中的第二个酪氨酸被磷酸化时，该肽对dNumb PTB结构域的亲和力增加（Kd = 1.41 +/- 0.10微摩尔）。对磷酸肽的氨基酸取代研究表明，序列GP(p)Y的核心基序是与dNumb PTB结构域高亲和力结合所必需的。对与含酪氨酸或磷酸酪氨酸的肽复合的同位素标记蛋白质进行的核磁共振实验表明，dNumb PTB结构域中同一组氨基酸参与了肽的磷酸化和非磷酸化形式的结合。因此，dNumb PTB结构域的体外选择性与Shc和IRS-1 PTB结构域明显不同，因为它优先与GP(p)Y基序相互作用，而不是NPXpY，并且结合时并不绝对需要配体磷酸化。我们的结果表明，PTB结构域是一种通用的蛋白质模块，能够表现出不同的结合特异性。
