Binding Mechanism of the N-Terminal SH3 Domain of CrkII and Proline-Rich Motifs in cAbl

Abstract

The N-terminal Src homology 3 (nSH3) domain of a signaling adaptor protein, CT-10 regulator of kinase II (CrkII), recognizes proline-rich motifs (PRMs) of binding partners, such as cAbl kinase. The interaction between CrkII and cAbl kinase is involved in the regulation of cell spreading, microbial pathogenesis, and cancer metastasis. Here, we report the detailed biophysical characterizations of the interactions between the nSH3 domain of CrkII and PRMs in cAbl. We identified that the nSH3 domain of CrkII binds to three PRMs in cAbl with virtually identical affinities. Structural studies, by using x-ray crystallography and NMR spectroscopy, revealed that the binding modes of all three nSH3:PRM complexes are highly similar to each other. Van 't Hoff analysis revealed that nSH3:PRM interaction is associated with favorable enthalpy and unfavorable entropy change. The combination of experimentally determined thermodynamic parameters, structure-based calculations, and (15)N NMR relaxation analysis highlights the energetic contribution of conformational entropy change upon the complex formation, and water molecules structured in the binding interface of the nSH3:PRM complex. Understanding the molecular basis of nSH3:PRM interaction will provide, to our knowledge, new insights for the rational design of small molecules targeting the interaction between CrkII and cAbl.

Atomic view of the energy landscape in the allosteric regulation of Abl kinase

“…Src 49 ). Crk, a scaffold protein consisting of a tandem SH2-SH3-SH3, binds through its middle SH3 domain to the proline-rich Crk-binding sites located at the disordered C-terminal tail of Abl 50,51 (Fig. 1a) and uses its SH2 domain to bind to the linker SH2-KD (ref 52).…”

Molecular Mechanisms of Tight Binding through Fuzzy Interactions

“…All protein samples for crystallization, fluorescence, and NMR experiments were prepared as described elsewhere (12). Synthetic peptides were purchased in a crude form and further purified using reverse-phase highperformance liquid chromatography in our laboratory.…”

“…The binding affinity of the nSH3 domain with PRM NS1 is significantly higher than its interactions with other cellular binding partners. For example, the nSH3 domain binds PRM NS1 with $3000-fold higher affinity than the PRM of JNK1 (11,12). It was suggested that the high affinity is due to long-range electrostatic interactions between positively charged residues in PRM NS1 and a negatively charged binding interface in the nSH3 domain (11).…”

Optimizing the Amino Acid Sequences of Peptides and Improving Their Specificity of Binding to SH3 Domains of Target Proteins

“…The above results showed that promiscuous binding of peptide molecule can be effectively ameliorated by rational design. Based on various public crystallographic data, [33][34][35][36][37][38] the SH3 domain has been thoroughly researched and broadly recognized as one of the best available systems for the examination of ligand-protein interactions. For example, Larson et al constructed a diverse alignment of SH3 domain sequences.…”