Arg-14 loop of site 3 anemone toxins: effects of glycine replacement on toxin affinity.
Anthopleurin B (ApB) is a high-affinity sea anemone neurotoxin that interacts with voltage-sensitive sodium (Na(V)) channels, causing a delay in channel inactivation. The solution structures of all known anemone toxins having this activity include a poorly defined region encompassing ApB residues 8-17, which we call the Arg-14 loop. We propose that the inherent mobility of the Arg-14 loop is necessary for the toxins' ability to maintain a high-affinity channel complex throughout the continual conformational transitions experienced by the channel during its functional cycle. We have previously shown that Arg-12, located in this loop, and Leu-18, which is adjacent, are important for ApB activity. Here, we characterized the role of two glycines located within the loop (Gly-10 and Gly-15) and an additional glycine positioned immediately C-terminal to it (Gly-20). We used site-directed replacement by alanine to assess the functional contribution to toxin binding of each of these residues singly and in combination. Gly-20 was found to be an essential toxin folding determinant; Gly-10 and Gly-15 were important for determining toxin affinity. Compared to wild-type toxin, the G10A and G15A toxins displayed significantly higher K(D) values for both cardiac (Na(V)1.5) and neuronal (Na(V)1.2) channels, although both demonstrated greater isoform discrimination for Na(V)1.5 than did wild-type ApB. For both G10A and G15A, significant Na(V) isoform differences were evident for on- and off-rates, with the most dramatic effect of a single mutation being the 467-fold reduction in the on-rate for G10A binding to Na(V)1.2, suggestive of a more accommodating binding site on Na(V)1.5 as compared to Na(V)1.2. Because alanine replacement of glycines is known to be associated with reduced backbone freedom, these results suggest an essential role for Arg-14 loop flexibility in toxin function, although a direct steric effect of the mutant methyl group cannot be excluded.
Seibert
Anna L
AL
Department of Biochemistry, State University of New York at Buffalo, Buffalo, New York 14214, USA.
Liu
Jinrong
J
Hanck
Dorothy A
DA
Blumenthal
Kenneth M
KM
D000409:Alanine / Q000235:genetics; D019943:Amino Acid Substitution / Q000235:genetics*; D000818:Animals; D001120:Arginine / Q000235:genetics* / Q000378:metabolism; D001665:Binding Sites / Q000235:genetics; D002460:Cell Line; D045744:Cell Line, Tumor; D003064:Cnidarian Venoms / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D005998:Glycine / Q000235:genetics* / Q000378:metabolism; D006801:Humans; D036341:Intercellular Signaling Peptides and Proteins; D007700:Kinetics; D051379:Mice; D008958:Models, Molecular; D009124:Muscle Proteins / Q000378:metabolism; D016297:Mutagenesis, Site-Directed; D062551:NAV1.2 Voltage-Gated Sodium Channel; D062554:NAV1.5 Voltage-Gated Sodium Channel; D009419:Nerve Tissue Proteins / Q000378:metabolism; D018408:Patch-Clamp Techniques; D010455:Peptides / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D017434:Protein Structure, Tertiary / Q000235:genetics; D015222:Sodium Channels / Q000378:metabolism
D003064:Cnidarian Venoms; D036341:Intercellular Signaling Peptides and Proteins; D009124:Muscle Proteins; D062551:NAV1.2 Voltage-Gated Sodium Channel; D062554:NAV1.5 Voltage-Gated Sodium Channel; D009419:Nerve Tissue Proteins; D010455:Peptides; C568251:SCN2A protein, human; C568320:SCN5A protein, human; C000654500:Scn2a protein, mouse; C568321:Scn5a protein, mouse; D015222:Sodium Channels; C026132:anthopleurin B; D001120:Arginine; D000409:Alanine; D005998:Glycine