Selective photoaffinity labeling of the inositol polyphosphate binding C2B domains of synaptotagmins

Synaptotagmin (Syt) II, a synaptic vesicle protein containing two copies of highly conserved protein kinase C homology regions known as the C2A and C2B domains, acts as a Ca²⁺ sensor and provides both phospholipid and inositol polyphosphate (IP(n)) recognition domains important in ends- and exocytosis. Four photoaffinity analogues of IP₃, IP₄, and IP₆ containing a P-1- or P-2-linked 4-benzoyidihydrocinnamidyl (BZDC) photophore were used to label glutathione S-transferase (GST) fusion constructs of the Syt II-C2A and C2B domains. The P-2-linked [³H]BZDC-IP₆ showed efficient, IP₆- displaceable labeling of the GST-Syt II-C2B. The rank order of photocovalent modification paralleled the order of competitive displacement: IP₆ (P-2- 1inked) > IP₄ > IP₃. The P-1-linked [³H]BZDC-IP₆ failed to label the C2B domains. The GST-Syt III-C2B domain, which lacks IP₆ binding affinity, also failed to undergo labeling by P-2-linked [³H]BZDC-IP₆. When mixtures of the 32-amino acid basic peptide corresponding to the essential IP(n) binding region of the Syt II-C2B domain and GST-Syt II-C2B were labeled by a stoichiometric amount of P-2-linked [³H]BZDC-IP₆, the two polypeptides showed equivalent affinity for the photolabel. Although the CD spectrum of this 32-mer at two pH values showed a random coil, the photoaffinity analogue of IP₆ appeared to induce a binding-compatible structure in the short peptide.