Delineation of the functional site of a snake venom cardiotoxin: preparation, structure, and function of monoacetylated derivatives.
Toxin gamma, a cardiotoxin from the venom of the cobra Naja nigricollis, was modified with acetic anhydride, and the derivatives were separated by cation-exchange and reverse-phase chromatography. Nine monoacetylated derivatives were obtained, and those modified at positions 1, 2, 12, 23, and 35 were readily identified by automated sequencing. The overall structure of toxin gamma, composed of three adjacent loops (I, II, and III) rich in beta-sheet, was not affected by monoacetylation as revealed by circular dichroic analysis. Trp-11, Tyr-22, and Tyr-51 fluorescence intensities were not affected by modifications at Lys-12 and Lys-35, whereas Trp-11 fluorescence intensity slightly increased when Lys-1 and Lys-23 were modified. The cytotoxic activity of toxin gamma to FL cells in culture was unchanged after modification at positions 1 and 2, whereas it was 3-fold lower after modification at Lys-23 and Lys-35. The derivative modified at Lys-12 was 10-fold less active than native toxin. Using two isotoxins, we found that substitutions at positions 28, 30, 31, and 57 did not change the cytotoxic potency of toxin gamma. A good correlation between cytotoxicity, lethality, and, to some extent, depolarizing activity on cultured skeletal muscle cells was found. In particular, the derivative modified at Lys-12 always had the lowest potency. Our data show that the site responsible for cytotoxicity, lethality, and depolarizing activity is not diffuse but is well localized on loop I and perhaps at the base of loop II. This site is topographically different from the AcChoR binding site of the structurally similar snake neurotoxins.
Département de Biologie, CEN Saclay, Gif-sur-Yvette, France.
E
E
Gatineau
M
M
Takechi
F
F
Bouet
P
P
Mansuelle
H
H
Rochat
A L
AL
Harvey
T
T
Montenay-Garestier
A
A
Ménez
D000107:Acetylation; D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D002478:Cells, Cultured; D002942:Circular Dichroism; D004179:Cobra Cardiotoxin Proteins / Q000235:genetics / Q000633:toxicity; D005260:Female; D008564:Membrane Potentials / Q000187:drug effects; D051379:Mice; D008807:Mice, Inbred BALB C; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009132:Muscles / Q000187:drug effects; D011487:Protein Conformation; D012910:Snake Venoms; D013050:Spectrometry, Fluorescence; D013329:Structure-Activity Relationship
D004179:Cobra Cardiotoxin Proteins; D012910:Snake Venoms