The growth factor receptor-binding protein Grb2, composed of one SH2 and two SH3 domains, acts as an adaptor protein in transduction of signals from tyrosine-phosphorylaled proteins (EGF-receptor, or SHC), to the Ras dependent pathways. It binds to phosphotyrosine in the cytoplasmic tail of cell surface receptors via its central SH2 domain, and to its immediate downstream target. Sos (a Ras guaninc nucleotide releasing factor), via its SH3 domains. Here, we describe an approach called ''consolidated" ligands to probe structural and functional activities of multidomain proteins. These ligands, having multiple binding portions, may be expected to bind with high affinity and specificity. when a linker between segments is of correct length. Based on the structure of whole Grb2, consolidated ligands were designed and synthesized. The linkers here are made of glycyi moieties. The ligands with two binding portions can block simultaneously the one SH2 and one SH3, or two SH3 domains of Grb'2 with better than 100 nM affinity. A higher order consolidated ligand with three binding portions was developed and high affinity binding was achieved. This study extends the feasibility of consolidated ligands, and this general protocol can be used to identify the interfaces between domains for rational design of higher affinity reagents.
|Original language||English (US)|
|Publication status||Published - Dec 1 1998|
ASJC Scopus subject areas
- Molecular Biology