Structure-activity relationships in Kluyveromyces lactis γ-toxin, a eukaryal tRNA anticodon nuclease
Structure-activity relationships in Kluyveromyces lactis γ-toxin, a eukaryal tRNA anticodon nuclease
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DOI：
10.1261/rna.1637809
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发表时间：
2009-06-01
期刊：
影响因子：
4.5
通讯作者：
Shuman, Stewart
中科院分区：
文献类型：
--
作者：
Keppetipola, Niroshika;Jain, Ruchi;Shuman, Stewart
由分泌性核毒素（例如乳酸克鲁维酵母伽马毒素和细菌大肠杆菌素）造成的 tRNA 反密码子损伤是区分自身和非自身物种的基本先天免疫系统的基础。 γ-毒素（一种 232 个氨基酸的多肽）的细胞内表达通过切割 tRNA (Glu) 修饰的摆动碱基的单个 RNA 磷酸二酯 3' 来阻止酿酒酵母的生长。真菌伽马毒素与任何已知的核酸酶没有一级结构相似性，并且在蛋白质数据库中没有可能的同源物。为了深入了解 γ-毒素的机制，我们测试了 62 个碱性、酸性和极性氨基酸的丙氨酸突变对体内核毒素活性的影响。由此，我们鉴定了 22 个必需残基，包括 10 个赖氨酸、7 个精氨酸、3 个谷氨酸、1 个半胱氨酸和 1 个组氨酸（His209，γ-毒素中存在的唯一组氨酸）。结构-活性关系是从 44 个保守取代的影响中收集的。重组无标签伽马毒素（一种单体蛋白）在摆动尿苷的单个磷酸二酯 3' 处切割了与 tRNA (Glu) 的反密码子茎环相对应的寡核苷酸。反密码子核酸酶不依赖于金属。摆动尿苷的核糖 2'-H 和 2'-F 修饰消除了 RNA 裂解。将 His209 突变为丙氨酸、谷氨酰胺或天冬酰胺会消除核酸酶活性。我们提出，γ-毒素催化类似 RNase A 的酯交换反应，该反应依赖于 His209 和第二个非组氨酸侧链作为通用酸碱催化剂。
tRNA anticodon damage inflicted by secreted ribotoxins such as Kluyveromyces lactis gamma-toxin and bacterial colicins underlies a rudimentary innate immune system that distinguishes self from nonself species. The intracellular expression of gamma-toxin (a 232-amino acid polypeptide) arrests the growth of Saccharomyces cerevisiae by incising a single RNA phosphodiester 3' of the modified wobble base of tRNA(Glu). Fungal gamma-toxin bears no primary structure similarity to any known nuclease and has no plausible homologs in the protein database. To gain insight to gamma-toxin's mechanism, we tested the effects of alanine mutations at 62 basic, acidic, and polar amino acids on ribotoxin activity in vivo. We thereby identified 22 essential residues, including 10 lysines, seven arginines, three glutamates, one cysteine, and one histidine (His209, the only histidine present in gamma-toxin). Structure-activity relations were gleaned from the effects of 44 conservative substitutions. Recombinant tag-free gamma-toxin, a monomeric protein, incised an oligonucleotide corresponding to the anticodon stem-loop of tRNA(Glu) at a single phosphodiester 3' of the wobble uridine. The anticodon nuclease was metal independent. RNA cleavage was abolished by ribose 2'-H and 2'-F modifications of the wobble uridine. Mutating His209 to alanine, glutamine, or asparagine abolished nuclease activity. We propose that gamma-toxin catalyzes an RNase A-like transesterification reaction that relies on His209 and a second nonhistidine side chain as general acid-base catalysts.