Abstract:
Drk, a Drosophila homologue of human GRB2, interacts with Sevenless (Sev) receptor via its SH2 domain, while the N- and C-terminal SH3 domains (Drk-NSH3 and Drk-CSH3, respectively) are responsible for the interaction with proline-rich motifs (PRMs) of Son of sevenless (Sos) or Daughter of Sevenless (Dos). Drk-NSH3 on its own has a conformational equilibrium between folded and unfolded states, and the folded state is stabilised by the association with a Sos-derived proline-rich peptide with PxxPxR motif. In contrast, Drk-CSH3 is supposed to bind PxxxRxxKP motifs in Dos. Aiming at clarifying the structural and functional differences between the two SH3 domains, we performed NMR studies of Drk-CSH3. The resulting solution structure and the 15N-relaxation data showed that Drk-CSH3 consists of a stable domain. Large chemical shift perturbation was commonly found around the RT loop and the hydrophobic patch, while there were also changes that occur characteristically for Sos- or Dos-derived peptides. Sos-derived two peptides with PxxPxR motif showed stronger affinity to Drk-CSH3, indicating that the Sos PRMs can bind both N- and C-SH3 domains. Dos-derived two peptides could also bind Drk-CSH3, but with much weaker affinity, suggesting a possibility that any cooperative binding of Dos-PRMs may strengthen the Drk-Dos interaction. The NMR studies as well as the docking simulations provide valuable insights into the biological and biophysical functions of two SH3 domains in Drk.
摘要:
Drk,人类GRB2的果蝇同源物,通过其SH2结构域与Sevenless(Sev)受体相互作用,而N端和C端SH3结构域(分别为Drk-NSH3和Drk-CSH3)负责与七子(Sos)或七子(Dos)的富含脯氨酸的基序(PRM)的相互作用。Drk-NSH3本身在折叠状态和展开状态之间具有构象平衡,并且通过与具有PxxPxR基序的Sos衍生的富含脯氨酸的肽缔合来稳定折叠状态。相比之下,Drk-CSH3应该在Dos中绑定PxxxRxxKP主题。为了阐明两个SH3结构域之间的结构和功能差异,我们进行了Drk-CSH3的NMR研究。所得的溶液结构和15N-弛豫数据表明Drk-CSH3由稳定的结构域组成。大的化学位移扰动通常在RT环和疏水斑块周围发现,而So-或Dos-衍生的肽也有特征性的变化。具有PxxPxR基序的Sos衍生的两个肽对Drk-CSH3显示出更强的亲和力,表明SosPRM可以结合N-和C-SH3结构域。Dos衍生的两种肽也可以结合Drk-CSH3,但亲和力较弱,这表明Dos-PRM的任何合作结合都可能加强Drk-Dos相互作用。NMR研究以及对接模拟为Drk中两个SH3域的生物学和生物物理功能提供了宝贵的见解。