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SASH1 is a novel binding partner to disassemble Caskin1 tandem SAM homopolymer through heterogeneous SAM-SAM
Yanhui Wang1, Qiangou Chen1, Cang Wu2
1Shenzhen Key Laboratory for Neuronal Structural Biology, Biomedical Research Institute, Shenzhen Peking University-The Hong Kong University of Science and Technology Medical Center, China.
Abstract:
Calcium/calmodulin-dependent serine protein kinase (CASK) interaction protein 1/2 (Caskin1/2) is essential neuronal synaptic scaffold protein in nervous system development. Knockouts of Caskin1/2 display severe deficits in novelty recognition and spatial memory. The tandem sterile alpha motif (SAM) domains of Caskin1/2, also conserved in their Drosophila homolog Ckn, are known to form homopolymers, yet their dynamic regulation mechanism remains unclear. In this study, SAM and SH3 domain-containing protein 1 (SASH1) was first identified as a novel binding partner of Caskin1/2 through yeast two-hybrid (Y2H) screening. The SAM-SAM interaction between SASH1 and Caskin1 was biochemically characterized by size-exclusion chromatography (SEC), isothermal titration calorimetry (ITC), and glutathione-S-transferase (GST) pull-down and co-immunoprecipitation (co-IP) assays. Structural insights from AlphaFold2-predicted models of the Caskin1-SAMs/SASH1-SAM1 complex, along with mutagenesis validations, revealed key residues at the end-helix (EH)/mid-loop (ML) interface for this interaction. More interestingly, the Caskin1-SAMs homopolymer can be disrupted by the SAM-SAM interaction, which was consistently verified by using sedimentation, transmission electron microscopy (TEM), and immunofluorescence (IF) staining in heterologous cell lines. In summary, our findings provide a solid biochemical basis for the Caskin1/SASH1 interaction and propose a potential mechanism for regulating Caskin1/2 homopolymerization via SAM-SAM interactions. More importantly, the principle governing SAM homopolymer depolymerization is generalized via suggesting two distinct types of heterogeneous SAM-SAM interactions, offering fresh insights into SAM domain-mediated homopolymerization and depolymerization.
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