Site-directed mutagenesis of m1-toxin1: two amino acids responsible for stable toxin binding to M(1) muscarinic receptors.
m1-Toxin1 binds specifically and irreversibly to M(1) muscarinic receptors and can slow the dissociation of [(3)H]N-methylscopolamine ([(3)H]NMS) from these receptors. Yet only 7 of its 65 amino acids are not conserved in six other mamba toxins that bind reversibly to M(2)-M(5) muscarinic receptors. Two of these seven residues (Phe(38), Lys(65)) were mutated to corresponding residues of the other toxins (Ile(38), Glu(65)), to evaluate amino acids in m1-toxin1 that confer its remarkable affinity and specificity. The cDNA for m1-toxin1 was cloned from venom gland mRNA using polymerase chain reaction (PCR)-based techniques. Its nucleotide sequence is remarkably similar to those of other short-chain neurotoxins. The cDNAs for mutant toxins Phe(38) to Ile(38) (F38I) and Lys(65) to Glu(65) (K65E) were constructed by PCR-based techniques. Each cDNA was expressed in yeast, and the toxins were purified from yeast media by cation-exchange and reversed phase chromatography. Recoveries were 40 to 152 microg/l. Recombinant m1-toxin1 was identical to the native toxin (observed mass: 7471 Da; irreversible blockade of [(3)H]NMS binding to cloned M(1) receptors at 25 degrees C; no blockade of M(2)-M(5) receptors; 6-fold slowing of [(3)H]NMS dissociation at 37 degrees C). F38I also bound specifically to M(1) receptors, but reversibly and without effect on NMS dissociation. Thus, Phe(38) contributes to the stability of toxin-receptor complexes, but not to M(1)-selectivity. K65E bound selectively and irreversibly to unliganded M(1) receptors but did not slow NMS dissociation. It is suggested that the C-terminal Lys(65) of m1-toxin1 may contact an outer loop of the M(1) receptor.
Krajewski
J L
JL
Department of Molecular and Cellular Pharmacology, University of Miami School of Medicine, Miami, Florida 33101, USA.
Dickerson
I M
IM
Potter
L T
LT
D000494:Allosteric Regulation; D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D003001:Cloning, Molecular; D018076:DNA, Complementary / Q000032:analysis; D004546:Elapid Venoms / Q000737:chemistry* / Q000235:genetics / Q000494:pharmacology*; D017815:Elapidae; D018698:Glutamic Acid / Q000235:genetics; D007532:Isoleucine / Q000235:genetics; D008239:Lysine / Q000235:genetics; D008958:Models, Molecular; D008969:Molecular Sequence Data; D018727:Muscarinic Antagonists / Q000494:pharmacology*; D016297:Mutagenesis, Site-Directed; D010649:Phenylalanine / Q000235:genetics; D043643:Receptor, Muscarinic M1; D011976:Receptors, Muscarinic / Q000187:drug effects / Q000235:genetics / Q000378:metabolism*; D011994:Recombinant Proteins / Q000494:pharmacology; D017386:Sequence Homology, Amino Acid
D018076:DNA, Complementary; D004546:Elapid Venoms; D018727:Muscarinic Antagonists; D043643:Receptor, Muscarinic M1; D011976:Receptors, Muscarinic; D011994:Recombinant Proteins; C083667:m1-toxin; D007532:Isoleucine; D018698:Glutamic Acid; D010649:Phenylalanine; D008239:Lysine