Flexibility of interdomain contacts revealed by topological isomers of bivalent consolidated ligands to the dual Src homology domain SH(32) of Abelson

Src homology (SH)2 and SH3 domains are found in a variety of proteins invol ved in the control of cellular signaling and architecture. The possible int errelationships between domains are not easily investigated, even though se veral cases of multiple domain-containing constructs have been studied stru cturally. As a complement to direct structural methods, we have developed c onsolidated ligands and tested their binding to the Abl SH(32) complex. Con solidated 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 lin kers. Four types of ligands were chemically synthesized, representing all t he possible relative orientations of ligands, Their affinities were found t o vary with binding portion topologies and linker lengths. Two of these typ es were shown to bind to both SH2 and SH3 dual domains with high affinities and specificities, showing increases of one order of magnitude, as compare d to the most strongly bound monovalent equivalent. These results suggest t hat 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 struct ures of ligated complexes and for developing reagents with high affinities and specificities.