Mutational analysis of the ligand binding site of the inositol 1,4,5-trisphosphate receptor.
To define the structural determinants for inositol 1,4, 5-trisphosphate (IP3) binding of the type 1 inositol 1,4, 5-trisphosphate receptor (IP3R1), we developed a means of expressing the N-terminal 734 amino acids of IP3R1 (T734), which contain the IP3 binding region, in Escherichia coli. The T734 protein expressed in E. coli exhibited a similar binding specificity and affinity for IP3 as the native IP3R from mouse cerebellum. Deletion mutagenesis, in which T734 was serially deleted from the N terminus up to residue 215, markedly reduced IP3 binding activity. However, when deleted a little more toward the C terminus (to residues 220, 223, and 225), the binding activity was retrieved. Further N-terminal deletions over the first 228 amino acids completely abolished it again. C-terminal deletions up to residue 579 did not affect the binding activity, whereas those up to residue 568 completely abolished it. In addition, the expressed 356-amino acid polypeptide (residues 224-579) exhibited specific binding activity. Taken together, residues 226-578 were sufficient and close enough to the minimum region for the specific IP3 binding, and thus formed an IP3 binding "core." Site-directed mutagenesis was performed on 41 basic Arg and Lys residues within the N-terminal 650 amino acids of T734. We showed that single amino acid substitutions for 10 residues, which were widely distributed within the binding core and conserved among all members of the IP3R family, significantly reduced the binding activity. Among them, three (Arg-265, Lys-508, and Arg-511) were critical for the specific binding, and Arg-568 was implicated in the binding specificity for various inositol phosphates. We suggest that some of these 10 residues form a basic pocket that interacts with the negatively charged phosphate groups of IP3.
Yoshikawa
F
F
Department of Molecular Neurobiology, Institute of Medical Science, University of Tokyo, Japan.
Morita
M
M
Monkawa
T
T
Michikawa
T
T
Furuichi
T
T
Mikoshiba
K
K
D000595:Amino Acid Sequence; D000818:Animals; D001120:Arginine / Q000235:genetics; D001483:Base Sequence; D001665:Binding Sites; D015220:Calcium Channels / Q000235:genetics / Q000378:metabolism*; D004252:DNA Mutational Analysis; D004926:Escherichia coli / Q000235:genetics; D005973:Glutamine / Q000235:genetics; D015544:Inositol 1,4,5-Trisphosphate / Q000378:metabolism*; D053496:Inositol 1,4,5-Trisphosphate Receptors; D008024:Ligands; D008239:Lysine / Q000235:genetics; D051379:Mice; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010446:Peptide Fragments / Q000378:metabolism; D018160:Receptors, Cytoplasmic and Nuclear / Q000235:genetics / Q000378:metabolism*; D011994:Recombinant Proteins / Q000378:metabolism; D017384:Sequence Deletion; D017386:Sequence Homology, Amino Acid; D013329:Structure-Activity Relationship
D015220:Calcium Channels; D053496:Inositol 1,4,5-Trisphosphate Receptors; D008024:Ligands; D010446:Peptide Fragments; D018160:Receptors, Cytoplasmic and Nuclear; D011994:Recombinant Proteins; D005973:Glutamine; D015544:Inositol 1,4,5-Trisphosphate; D001120:Arginine; D008239:Lysine