Simon J A, Schreiber S L
Howard Hughes Medical Institute, Cambridge, MA, USA.
Chem Biol. 1995 Jan;2(1):53-60. doi: 10.1016/1074-5521(95)90080-2.
Grb2 acts as an adaptor protein in the transduction of signals from receptor tyrosine kinases to Ras. It binds to phosphotyrosine on the cytoplasmic tail of cell-surface receptors via its central SH2 domain, and to its immediate downstream target, Sos, via two SH3 domains. The basis of the Grb2-Sos interaction is not fully understood. We previously proposed a model for SH3 domain binding specificity, based on two solution structures of the Src SH3 domain complexed with high-affinity ligands, in which the ligands are bound in a polyproline type II conformation in two distinct orientations, class I and class II. Here, we have used this model to predict the identity and orientation of Grb2 SH3 ligands in the human Sos protein.
Six contiguous fragments from the carboxy-terminal portion of hSos (amino acids 1000-1333), each containing a single potential SH3 binding site, were expressed in E. coli as GST fusion proteins. Four of these proteins were predicted to associate with SH3 domains. The amino-terminal Grb2 SH3 domain was shown to bind strongly to only these four fragments.
We have used a general model for SH3-ligand interactions to predict the nature of Grb2 SH3 interactions with the hSos protein. Comparison of the four hSos sequences that bind Grb2 revealed a preference for the PXXPXR motif, consistent with the predicted class II-type binding interaction. The interaction between Grb2 and hSos peptides is predominantly via the amino-terminal SH3 domain, although the carboxy-terminal SH3 domain may increase the overall stability of the Grb2-hSos complex.
Grb2在从受体酪氨酸激酶到Ras的信号转导中作为衔接蛋白发挥作用。它通过其中心的SH2结构域与细胞表面受体胞质尾部的磷酸酪氨酸结合，并通过两个SH3结构域与它的直接下游靶点Sos结合。Grb2-Sos相互作用的基础尚未完全了解。我们之前基于Src SH3结构域与高亲和力配体复合的两个溶液结构提出了一个SH3结构域结合特异性模型，其中配体以两种不同的取向，即I类和II类，以多聚脯氨酸II型构象结合。在这里，我们使用这个模型来预测人Sos蛋白中Grb2 SH3配体的身份和取向。
来自hSos羧基末端部分（氨基酸1000 - 1333）的六个连续片段，每个片段包含一个潜在的SH3结合位点，在大肠杆菌中作为GST融合蛋白表达。预计这些蛋白中的四个与SH3结构域相关。氨基末端的Grb2 SH3结构域被证明仅与这四个片段强烈结合。
我们使用了一个SH3-配体相互作用的通用模型来预测Grb2 SH3与hSos蛋白相互作用的性质。对结合Grb2的四个hSos序列的比较揭示了对PXXPXR基序的偏好，这与预测的II类结合相互作用一致。Grb2与hSos肽之间的相互作用主要通过氨基末端的SH3结构域，尽管羧基末端的SH3结构域可能会增加Grb2-hSos复合物的整体稳定性。
