A biophysical and molecular characterization of the interaction between the Alzheimer risk factor BIN1 and the neuronal scaffold protein p140Cap

Bridging integrator 1 (BIN1) is a genetic risk factor for late-onset Alzheimer disease. BIN1's participation in endocytosis, membrane remodeling, and modulation of actin dynamics is well-characterized in non-neuronal cells. In neurons, BIN1 is enriched at presynaptic sites, where it facilitates excitatory neurotransmitter vesicle release. However, how BIN1 is involved in synaptic vesicle dynamics is not well understood. A C-terminal Src homology 3 (SH3) domain is invariant in all BIN1 isoforms and promotes protein-protein interactions with proteins harboring proline-rich motifs. While BIN1 interactions with dynamin, synaptojanin, RIN3, and tau have been identified and experimentally validated, the list of BIN1-interacting molecules is not exhaustive. Here, we report the neuronal scaffolding protein p140Cap, encoded by SRC kinase signaling inhibitor 1, as a BIN1 SH3 domain-interacting protein. We performed surface plasmon resonance to ascertain the affinity of BIN1-SH3 domain for p140Cap and identified a peptide containing three proline-rich motifs that exhibited biologically relevant affinity (K_D = 7.7 μM). Additional surface plasmon resonance experiments, coupled with alanine-scanning mutagenesis, revealed that two class II motifs, but not a class I motif, in p140Cap facilitated binding. Confocal microscopy and proximity ligation assays confirmed that BIN1 colocalizes with, and is within molecular distance of, p140Cap in cultured cells and in the mouse brain. Coimmunoprecipitation assays validated the interaction and glutathione S-transferase pulldown revealed that a rare BIN1 coding variant (rs138047593) significantly reduces p140Cap and tau binding, highlighting the impact of this mutant on interacting protein binding efficiency. The functional implications of BIN1:p140Cap interaction for neuronal functions warrant further investigation.