Characterization of saxitoxin binding to saxiphilin, a relative of the transferrin family that displays pH-dependent ligand binding.
Saxiphilin is a 91 kDa saxitoxin-binding protein that is homologous to members of the transferrin family of Fe(3+)-binding proteins noted for pH-dependent release of Fe3+. The mechanism of toxin binding to purified native saxiphilin from the bullfrog (Rana catesbeiana) was studied using [3H]saxitoxin. At pH 7.4 and 0 degrees C [3H]saxitoxin binds to a single site on saxiphilin with a KD of approximately 0.2 nM. The pH dependence of [3H]saxitoxin binding follows a one-site titration curve in the range of pH 9-4 with maximal binding from pH 9 to 7 and half-inhibition at pH 5.7. Inhibition of toxin binding at low pH is the combined result of a decrease in the rate of toxin association and an increase in the rate of toxin dissociation. The dependence of the apparent rate constants for [3H]saxitoxin association and dissociation on [H+] can be accounted for by a four-state model of allosteric interaction between the toxin-binding site and a single titratable residue of saxiphilin with a pKa of 7.2 in the toxin-free form and 4.3 in the toxin-bound form. From 0 to 25 degrees C, the temperature dependence of [3H]saxitoxin binding to saxiphilin is characterized by delta H degrees = -8.3 kcal mol-1, delta S degrees = 13.8 cal mol-1 K-1, and activation energies of 22.5 kcal mol-1 for dissociation and 11.1 kcal mol-1 for association. Binding of [3H]saxitoxin to saxiphilin is competitively inhibited with low affinity by a variety of divalent metal and lanthanide cations. Inhibition of toxin binding by the carboxyl-methylating reagent trimethyloxonium is prevented by pre-equilibration with [3H]saxitoxin, implicating the presence of one or more carboxyl groups in the binding site. Functional similarities suggest that the saxitoxin-binding site of saxiphilin is located in an interdomain cleft analogous to the location of one of the two homologous Fe(3+)-binding sites of transferrins. On the basis of residue substitutions between saxiphilin and transferrins, it is proposed that the saxitoxin-binding site is located in the carboxy terminal lobe of saxiphilin and that binding is modulated by protonation of a conserved histidine residue.
Llewellyn
L E
LE
Department of Pharmacology, Yale University School of Medicine, New Haven, Connecticut 06520-8066.
Moczydlowski
E G
EG
D000595:Amino Acid Sequence; D029845:Amphibian Proteins; D000818:Animals; D001665:Binding Sites / Q000187:drug effects; D002352:Carrier Proteins; D004591:Electrophoresis, Polyacrylamide Gel; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008024:Ligands; D008969:Molecular Sequence Data; D009861:Onium Compounds / Q000494:pharmacology; D011892:Rana catesbeiana; D012530:Saxitoxin / Q000378:metabolism*; D015222:Sodium Channels / Q000737:chemistry / Q000378:metabolism*; D013696:Temperature
D029845:Amphibian Proteins; D002352:Carrier Proteins; D008024:Ligands; D009861:Onium Compounds; D015222:Sodium Channels; C027525:saxitoxin-binding protein, Rana catesbeiana; D012530:Saxitoxin; C009576:trimethyloxonium