Tira Roberto, Leo Giulia, Prandini Laura, Barracchia Carlo Giorgio, D'Onofrio Mariapina, Mollica Luca, Capaldi Stefano, Assfalg Michael, Munari Francesca
Department of Biotechnology, University of Verona, Verona, Italy.
Department of Medical Biotechnology and Translational Medicine, University of Milan, Milan, Italy.
Angew Chem Int Ed Engl. 2025 Jul 7;64(28):e202504292. doi: 10.1002/anie.202504292. Epub 2025 May 23.
The interaction between the microtubule associated protein Tau and the tyrosine kinase Fyn is believed to play a pivotal role in the early stage of Alzheimer's disease. Previous studies have identified the SRC Homology 3 (SH3) domain of Fyn as the binding receptor of several proline-rich motifs in Tau. However, the role of each proline-rich motif and their interplay in molecular recognition are still unclear. In this work, we investigated the mechanism of Fyn-SH3 recognition by the multiple PxxP sites inserted within the full-length Tau protein by using nuclear magnetic resonance (NMR) spectroscopy combined with computational, calorimetric and in-cell FRET (Förster resonance energy transfer) methods. Both in vitro and in-cell experiments revealed no single binding site strictly necessary for the binding. Instead, Fyn-SH3 contacts full-length Tau on multiple hot spot regions, located over a distance of 85 residues, through global moderate-to-low affinity interactions. Beyond two principal regions containing classical PxxP motifs, we identified a novel non-canonical binding site at the beginning of the microtubule binding domain. Our study indicates that multiple binding sites in Tau are involved in the interaction, making Tau an adaptable recognition surface that can function when single consensus motifs are deleted.
微管相关蛋白Tau与酪氨酸激酶Fyn之间的相互作用被认为在阿尔茨海默病的早期阶段起着关键作用。先前的研究已确定Fyn的SRC同源3（SH3）结构域是Tau中几个富含脯氨酸基序的结合受体。然而，每个富含脯氨酸基序的作用及其在分子识别中的相互作用仍不清楚。在这项工作中，我们通过使用核磁共振（NMR）光谱结合计算、量热法和细胞内荧光共振能量转移（FRET）方法，研究了全长Tau蛋白中插入的多个PxxP位点对Fyn-SH3的识别机制。体外和细胞内实验均表明，不存在对结合严格必需的单个结合位点。相反，Fyn-SH3通过全局中等到低亲和力相互作用，在多个热点区域与全长Tau接触，这些区域跨越85个残基的距离。除了包含经典PxxP基序的两个主要区域外，我们在微管结合结构域的起始处鉴定出一个新的非经典结合位点。我们的研究表明，Tau中的多个结合位点参与了这种相互作用，使Tau成为一个适应性的识别表面，当单个共有基序缺失时仍能发挥作用。
