Site-directed mutagenesis of m1-toxin1: two amino acids responsible for stable toxin binding to M(1) muscarinic receptors.
Site-directed mutagenesis of m1-toxin1: two amino acids responsible for stable toxin binding to M(1) muscarinic receptors.
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DOI：
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发表时间：
2001-10
影响因子：
3.6
通讯作者：
J. Krajewski;I. Dickerson;L. Potter
中科院分区：
文献类型：
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作者：
J. Krajewski;I. Dickerson;L. Potter
m1-Toxin1 特异性且不可逆地与 M(1) 毒蕈碱受体结合，并可以减缓 [(3)H]N-甲基东莨菪碱 ([(3)H]NMS) 从这些受体上的解离。然而，其 65 个氨基酸中只有 7 个在与 M(2)-M(5) 毒蕈碱受体可逆结合的其他六种曼巴毒素中不保守。这七个残基中的两个 (Phe(38)、Lys(65)) 突变为其他毒素的相应残基 (Ile(38)、Glu(65))，以评估 m1-toxin1 中赋予其显着亲和力和特异性的氨基酸。 m1-toxin1 的 cDNA 是使用基于聚合酶链反应 (PCR) 的技术从毒腺 mRNA 中克隆的。其核苷酸序列与其他短链神经毒素的核苷酸序列非常相似。通过基于 PCR 的技术构建了突变毒素 Phe(38) 至 Ile(38) (F38I) 和 Lys(65) 至 Glu(65) (K65E) 的 cDNA。每个 cDNA 在酵母中表达，并通过阳离子交换和反相色谱从酵母培养基中纯化毒素。回收率为 40 至 152 微克/升。重组 m1-toxin1 与天然毒素相同（观察到的质量：7471 Da；在 25 摄氏度下不可逆地阻断 [(3)H]NMS 与克隆的 M(1) 受体的结合；对 M(2)-M(5) 受体没有阻断；在 37 摄氏度下 [(3)H]NMS 解离减慢 6 倍）。 F38I 还特异性结合 M(1) 受体，但可逆地结合并且不影响 NMS 解离。因此，Phe(38) 有助于毒素-受体复合物的稳定性，但不有助于 M(1)-选择性。 K65E 选择性且不可逆地与未配体的 M(1) 受体结合，但不会减缓 NMS 解离。 m1-toxin1 的 C 端 Lys(65) 可能与 M(1) 受体的外环接触。
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.