Conformation-Driven Phase Separation in the LinkerDomain of Focal Adhesion Kinases

Protein tyrosine kinase 2 (Pyk2), also known as focal adhesion kinase 2, and focal adhesion kinase 1 (Fak1) are two related nonreceptor tyrosine kinases (hereafter referred to as FAKs). Here, we focused on characterizing a linker region of the FAK proteins (hereafter referred to as FAK KFL for Kinase FAT Linker), which in the case of Pyk2 has previously been shown to play a functional role in calcium sensing through its interaction with calmodulin. Using structural nuclear magnetic resonance spectroscopy, we provide chemical shift assignments for the FAK KFLs, defining their conformational properties. Analysis of the FAK KFL conformations revealed their predominantly disordered nature, except for well-defined segments with a significant tendency to form α-helices, which were modeled to form homodimeric interfaces. In addition, we showed that the FAK KFL segments form condensates in vitro under high crowding conditions. By directly comparing the conformational properties of the Pyk2 and Fak1 KFL domains and providing structural data, this study provides valuable insights into the structural basis of FAK KFL interactions. Furthermore, the results show that disordered segments in proteins within the focal adhesion complex undergo phase separation, a process of potential biological significance due to protein clustering.