Structure of a regulatory complex involving the Abl SH3 domain, the Crk SH2 domain, and a Crk-derived phosphopeptide

Abstract

On phosphorylation of Y221 by Abelson (Abl) kinase, the Crk-II adapter protein undergoes an intramolecular reorganization initiated by the binding of its own Src homology 2 (SH2) domain to the pY221 site. Conformational changes induced by phosphotyrosine recognition promote the binding of the Src homology 3 (SH3) domain of the Abl tyrosine kinase to a proline-rich loop located between the betaD and betaE strands of the SH2 domain (DE loop). We have determined the NMR solution structure of the ternary complex of the Abl SH3 domain with the Crk SH2 domain bound to a Crk pY221 phosphopeptide. The SH2 domain bridges two ligands that bind at distinct sites. The interaction between the Abl SH3 domain and the Crk SH2 domain is localized to a canonical eight-residue site within the DE loop. From (15)N relaxation experiments, the DE loop of the SH2 domain in the complex displays a significant degree of conformational freedom. The structural and dynamic data therefore indicate that these SH2 and SH3 domains do not assume a unique orientation with respect to one another; rather, they appear to be only tethered via the DE loop. Thus, SH2 domain-SH3 domain interactions do not require additional tertiary contacts or restriction of domain orientation when a recognition motif is presented in a mobile loop. This complex between the Abl SH3 domain, Crk SH2 domain, and Crk phosphopeptide is an example of the extremely modular nature of regulatory proteins that provides a rich repertoire of mechanisms for control of biological function.

Fig 1.

The complex described in this study includes the Abl SH3 domain (green), the Crk SH2 domain (yellow), and a 13-aa phosphopeptide corresponding to the Crk SH2 internal binding site (blue).

Fig 2.

Ternary complex of the Crk SH2 domain (yellow/orange, ribbon representation), Crk phosphopeptide (blue, heteroatom representation), and the Abl SH3 domain (green, ribbon representation). The loop in the Crk SH2 domain important for binding the Abl SH3 domain (DE loop) is labeled. Main-chain representation of the 20 lowest energy structures superposed according to the Abl SH3 domain (Upper Left) and the Crk SH2 domain/Crk phosphopeptide (Lower Right).

Fig 3.

Detail of the Abl SH3 domain bound to the Crk SH2 domain (residues 68–75) in the ternary complex (two orientations rotated 90° toward the viewer). Side chains of residues identified to make contacts to Crk SH2 domain are highlighted. D71 of Abl may participate in an ionic interaction with R68 of Crk. Secondary structure elements are labeled above the sequence, following the standard nomenclature for SH3 domains.

Fig 4.

(Upper) Detail of the Crk SH2 domain bound to the Crk phosphopeptide (residues 221–224) in the ternary complex. Residues observed to make contacts are highlighted. (Lower) Sequence comparison of Crk-II with homologues v-Crk and CrkL. The sequence of murine Crk-II corresponds to the protein fragment described in this study. Only Crk-II contains a binding site for the Abl SH3 domain (shaded box).