Dipartimento di Scienze Biochimiche "A. Rossi Fanelli", Laboratory affiliated to Istituto Pasteur Italia-Fondazione Cenci Bolognetti, Sapienza Università di Roma, Rome, Italy.
Protein Sci. 2023 Aug;32(8):e4729. doi: 10.1002/pro.4729.
In an effort to investigate the molecular determinants of ligand recognition of the C-terminal SH2 domain of the SHP2 protein, we conducted extensive site-directed mutagenesis and kinetic binding experiments with a peptide mimicking a specific portion of a physiological ligand (the scaffold protein Gab2). Obtained data provided an in-depth characterization of the binding reaction, allowing us to pinpoint residues topologically far from the binding pocket of the SH2 domain to have a role in the recognition and binding of the peptide. The presence of a sparse energetic network regulating the interaction with Gab2 was identified and characterized through double mutant cycle analysis, performed by challenging all the designed site-directed variants of C-SH2 with a Gab2 peptide mutated at +3 position relative to its phosphorylated tyrosine, a key residue for C-SH2 binding specificity. Results highlighted non-optimized residues involved in the energetic network regulating the binding with Gab2, which may be at the basis of the ability of this SH2 domain to interact with different partners in the intracellular environment. Moreover, a detailed analysis of kinetic and thermodynamic parameters revealed the role of the residue at +3 position on Gab2 in the early and late events of the binding reaction with the C-SH2 domain.
为了研究 SHP2 蛋白 C 末端 SH2 结构域与配体识别的分子决定因素，我们采用模拟生理配体（支架蛋白 Gab2）特定部分的肽进行了广泛的定点突变和动力学结合实验。获得的数据深入表征了结合反应，使我们能够确定拓扑上远离 SH2 结构域结合口袋的残基在识别和结合肽中的作用。通过双突变体循环分析鉴定并表征了存在稀疏的能量网络来调节与 Gab2 的相互作用，该分析通过用 Gab2 肽挑战所有设计的 C-SH2 定点突变体来进行，Gab2 肽在相对于其磷酸化酪氨酸的+3 位发生突变，该残基是 C-SH2 结合特异性的关键残基。结果突出了参与调节与 Gab2 结合的能量网络中非优化的残基，这可能是该 SH2 结构域在细胞内环境中与不同伴侣相互作用的基础。此外，对动力学和热力学参数的详细分析揭示了 Gab2 上+3 位残基在与 C-SH2 结构域的结合反应的早期和晚期事件中的作用。
