Crystallographic investigation of the dependence of calcium and phosphate ions for notexin.
The crystal structure of the neurotoxic phospholipase A2, notexin, revealed three binding sites for sulphate ions which were suggested to be phosphate binding sites of importance for the activity of the toxin. The present investigation shows that the sulphate ion bound to the major binding site alters the structure of residues 60-75. In the absence of sulphate and phosphate, the structure of this loop has a conformation which partly resembles the non-neurotoxic PLA2s. The affinity of notexin for phosphate is 17 microM, as measured by the increase in fluorescence at 345 nm. Since the concentrations of phosphate and sulphate ions in blood plasma are 3 and 1 mM, respectively, the binding site must be occupied under physiological conditions. This major sulphate/phosphate binding site explains the specific affinity labelling by pyridoxal phosphate. Pyridoxal phosphate binds to this anion binding site which allows the reaction with Lys-88 or Lys-89. The structure of notexin in the presence and absence of Ca2+ shows only small local structural differences.
Westerlund
B
B
Department of Biochemistry, Biomedical Centre, Uppsala, Sweden.
Saarinen
M
M
Person
B
B
Ramaswamy
S
S
Eaker
D
D
Eklund
H
H
D001665:Binding Sites; D002118:Calcium / Q000378:metabolism*; D018360:Crystallography, X-Ray; D004546:Elapid Venoms / Q000737:chemistry* / Q000378:metabolism*; D008958:Models, Molecular; D010710:Phosphates / Q000378:metabolism*; D011487:Protein Conformation; D011732:Pyridoxal Phosphate / Q000737:chemistry; D013431:Sulfates / Q000737:chemistry; D017186:Titrimetry
D004546:Elapid Venoms; D010710:Phosphates; D013431:Sulfates; C005082:notexin; D011732:Pyridoxal Phosphate; D002118:Calcium