Bok binds to a largely disordered loop in the coupling domain of type 1 inositol 1,4,5-trisphosphate receptor

Bcl-2-related ovarian killer (Bok) binds tightly to inositol 1,4,5-trisphosphate receptors (IP₃Rs). To better understand this interaction, we sought to elucidate the Bok binding determinants in IP₃R1, focusing on the ∼75 amino acid loop (residues 1882–1957) between α helices 72 and 73. Bioinformatic analysis revealed that the majority of this loop is intrinsically disordered, with two flanking regions of high disorder next to a low disorder central region (∼residues 1914–1926) that is predicted to contain two fused, disjointed transient helical elements. Experiments with IP₃R1 mutants, combined with computational analysis, indicated that small deletions in this central region block Bok binding due to perturbation of the helical elements. Studies in vitro with purified Bok and IP₃R1-derived peptides revealed high affinity binding to amino acids 1898–1940 of IP₃R1 (K_d ∼65 nM) and that binding affinity is also dependent upon both of the high disorder flanking regions. The strength of the Bok-IP₃R1 interaction was demonstrated by the ability of IP₃R1 or Bok to recruit transmembrane domain-free Bok or IP₃R1 mutants, respectively, to membranes in intact cells, and that these two mutants can bind in the cytosol independently of membrane association. Overall, we show that Bok binding to IP₃R1 occurs within a largely disordered loop between α helices 72 and 73 and that high affinity binding is mediated by multivalent interactions.