Siegal G, Davis B, Kristensen S M, Sankar A, Linacre J, Stein R C, Panayotou G, Waterfield M D, Driscoll P C
Ludwig Institute for Cancer Research, London, UK.
J Mol Biol. 1998 Feb 20;276(2):461-78. doi: 10.1006/jmbi.1997.1562.
Heterodimeric class IA phosphoinositide 3-kinase (PI 3-kinase) plays a crucial role in a variety of cellular signalling events downstream of a number of cell-surface receptor tyrosine kinases. Activation of the enzyme is effected in part by the binding of two Src homology-2 domains (SH2) of the 85 kDa regulatory subunit to specific phosphotyrosine-containing peptide motifs within activated cytoplasmic receptor domains. The solution structure of the uncomplexed C-terminal SH2 (C-SH2) domain of the p85 alpha subunit of PI 3-kinase has been determined by means of multinuclear, double and triple-resonance NMR experiments and restrained molecular-dynamics simulated-annealing calculations. The solution structure clearly indicates that the uncomplexed C-SH2 domain conforms to the consensus polypeptide fold exhibited by other SH2 domains, with an additional short helical element at the N terminus. In particular, the C-SH2 structure is very similar to both the p85 alpha N-terminal SH2 domain (N-SH2) and the Src SH2 domain with a root mean square difference (rmsd) for 44 C alpha atoms of 1.09 and 0.89 A, respectively. The canonical BC, EF and BG loops are less well-defined by the experimental restraints and show greater variability in the ensemble of C-SH2 conformers. The lower level of definition in these regions may reflect the presence of conformational disorder, an interpretation supported by the absence or broadening of backbone and side-chain NMR resonances for some of these residues. NMR experiments were performed, where C-SH2 was titrated with phosphotyrosine-containing peptides corresponding to p85 alpha recognition sites in the cytoplasmic domain of the platelet-derived growth-factor receptor. The ligand-induced chemical-shift perturbations indicate the amino-acid residues in C-SH2 involved in peptide recognition follow the pattern predicted from homologous complexes. A series of C-SH2 mutants was generated and tested for phosphotyrosine peptide binding by surface plasmon resonance. Mutation of the invariant Arg36 (beta B5) to Met completely abolishes phosphopeptide binding. Mutation of each of Ser38, Ser39 or Lys40 in the BC loop to Ala reduces the affinity of C-SH2 for a cognate phosphopeptide, as does mutation of His93 (BG5) to Asn. These effects are consistent with the involvement of the BC loop and BG loops regions in ligation of phosphopeptide ligands. Mutation of Cys57 (beta D5) in C-SH2 to Ile, the corresponding residue type in the p85 alpha N-SH2 domain, results in a change in peptide binding selectivity of C-SH2 towards that demonstrated by p85 alpha N-SH2. This pattern of p85 alpha phosphopeptide binding specificity is interpreted in terms of a model of the p85 alpha/PDGF-receptor interaction.
异二聚体IA类磷酸肌醇3激酶（PI 3激酶）在许多细胞表面受体酪氨酸激酶下游的各种细胞信号转导事件中起着关键作用。该酶的激活部分是通过85 kDa调节亚基的两个Src同源2结构域（SH2）与活化细胞质受体结构域内特定含磷酸酪氨酸的肽基序结合来实现的。PI 3激酶p85α亚基未结合的C末端SH2（C-SH2）结构域的溶液结构已通过多核、二维和三维共振NMR实验以及受限分子动力学模拟退火计算确定。溶液结构清楚地表明，未结合的C-SH2结构域符合其他SH2结构域呈现的共有多肽折叠，在N末端有一个额外的短螺旋元件。特别是，C-SH2结构与p85α N末端SH2结构域（N-SH2）和Src SH2结构域非常相似，44个Cα原子的均方根偏差（rmsd）分别为1.09和0.89 Å。实验限制对典型的BC、EF和BG环的定义不太明确，并且在C-SH2构象体集合中显示出更大的变异性。这些区域较低的定义水平可能反映了构象无序的存在，一些这些残基的主链和侧链NMR共振的缺失或变宽支持了这一解释。进行了NMR实验，用对应于血小板衍生生长因子受体细胞质结构域中p85α识别位点的含磷酸酪氨酸的肽滴定C-SH2。配体诱导的化学位移扰动表明，C-SH2中参与肽识别的氨基酸残基遵循从同源复合物预测的模式。生成了一系列C-SH2突变体，并通过表面等离子体共振测试其对磷酸酪氨酸肽的结合。不变的Arg36（βB5）突变为Met完全消除了磷酸肽结合。BC环中的Ser38、Ser39或Lys40中的每一个突变为Ala都会降低C-SH2对同源磷酸肽的亲和力，His93（BG5）突变为Asn也是如此。这些效应与BC环和BG环区域参与磷酸肽配体的连接一致。C-SH2中的Cys57（βD5）突变为p85α N-SH2结构域中的相应残基类型Ile，导致C-SH2对肽的结合选择性发生变化，向p85α N-SH2所示的方向变化。根据p85α/血小板衍生生长因子受体相互作用模型解释了p85α磷酸肽结合特异性的这种模式。
