Flexibility of Interdomain Contacts Revealed by Topological Isomers of Bivalent Consolidated Ligands to the Dual Src Homology Domain SH(32) of Abelson,

Abstract

Src homology (SH)2 and SH3 domains are found in a variety of proteins involved in the control of cellular signaling and architecture. The possible interrelationships between domains are not easily investigated, even though several cases of multiple domain-containing constructs have been studied structurally. As a complement to direct structural methods, we have developed consolidated ligands and tested their binding to the Abl SH(32) complex. Consolidated ligands combine in the same molecule peptide sequences recognized by SH2 and SH3 domains, i.e., Pro-Val-pTyr-Glu-Asn-Val and Pro-Pro-Ala-Tyr-Pro-Pro-Pro-Pro-Val-Pro, respectively; these are joined by oligoglycyl linkers. Four types of ligands were chemically synthesized, representing all the possible relative orientations of ligands. Their affinities were found to vary with binding portion topologies and linker lengths. Two of these types were shown to bind to both SH2 and SH3 dual domains with high affinities and specificities, showing increases of one order of magnitude, as compared to the most strongly bound monovalent equivalent. These results suggest that the relative orientation of SH2 and SH3 in Abl SH(32) is not fixed, and this synthetic approach may be generally useful for determining the structures of ligated complexes and for developing reagents with high affinities and specificities.