F. Yoshikawa et al., MUTATIONAL ANALYSIS OF THE LIGAND-BINDING SITE OF THE INOSITOL 1,4,5-TRISPHOSPHATE RECEPTOR, The Journal of biological chemistry, 271(30), 1996, pp. 18277-18284
To define the structural determinants for inositol 1,4,5-trisphosphate
(IP3) binding of the type 1 inositol 1,4,5-trisphosphate receptor (IP
(3)R1), we developed a means of expressing the N-terminal 734 amino ac
ids of IP(3)R1 (T734), which contain the IP3 binding region, in Escher
ichia coli. The T734 protein expressed in E. coli exhibited a similar
binding specificity and affinity for IP3 as the native IP(3)R from mou
se cerebellum. Deletion mutagenesis, in which T734 was serially delete
d from the N terminus up to residue 215, markedly reduced IP3 binding
activity. However, when deleted a little more toward the C terminus (t
o residues 220, 223, and 225), the binding activity was retrieved, Fur
ther N-terminal deletions over the first 228 amino acids completely ab
olished it again. C-terminal deletions up to residue 579 did not affec
t the binding activity, whereas those up to residue 568 completely abo
lished it. In addition, the expressed 356-amino acid polypeptide (resi
dues 224-579) exhibited specific binding activity. Taken together, res
idues 226-578 were sufficient and close enough to the minimum region f
or the specific IP3 binding, and thus formed an IP3 binding ''core.''
Site-directed mutagenesis was performed on 41 basic Arg and Lys residu
es within the N-terminal 650 amino acids of T734. We showed that singl
e amino acid substitutions for 10 residues, which were widely distribu
ted within the binding core and conserved among all members of the IP(
3)R family, significantly reduced the binding activity. Among them, th
ree (Arg-265, Lys-508, and Arg-511) were critical for the specific bin
ding, and Arg-568 was implicated in the binding specificity for variou
s inositol phosphates. We suggest that some of these 10 residues form
a basic pocket that interacts with the negatively charged phosphate gr
oups of IP3.