Structural determinants and cellular environment define processed actin as the sole substrate of the N-terminal acetyltransferase NAA80
Structural determinants and cellular environment define processed actin as the sole substrate of the N-terminal acetyltransferase NAA80
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DOI：
10.1073/pnas.1719251115
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发表时间：
2018-04-24
影响因子：
11.1
通讯作者：
Arnesen, Thomas
中科院分区：
文献类型：
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作者：
Goris, Marianne;Magin, Robert S.;Arnesen, Thomas
N-末端乙酰化是由N-末端乙酰转移酶（NATs）催化的蛋白质修饰的主要方式。在所有的真核生物中，包括肌动蛋白在内的甲基化酸性N末端都被NatB连续地Nt乙酰化，但是含有酸性N末端的动物肌动蛋白，也被NAA 80连续地Nt乙酰化。发现肌动蛋白Nt乙酰化调节细胞骨架动力学和运动性，从而使NAA 80成为细胞迁移调节的潜在靶点。在这项工作中，我们开发了有效的和选择性的双底物抑制剂的NAA 80和确定的晶体结构的NAA 80与这样的抑制剂的复合物，揭示了NAA 80采用类似于其他NAT酶的折叠，但具有更开放的底物结合区域。此外，与大多数其他NAT相反，NAA 80的底物特异性主要是通过酶与肌动蛋白底物的位置2和3而不是残基1和2处的酸性氨基酸之间的相互作用获得的。酵母模型显示，在缺乏NatB活性的菌株中异位表达NAA 80部分恢复了NatB底物（包括酵母肌动蛋白）的Nt乙酰化。因此，NAA 80在体内具有使Nt乙酰化NatB型底物的内在能力。总之，在所有的真核生物中存在一个占主导地位的共翻译NatB，在动物中特异性的翻译后肌动蛋白蛋氨酸去除，最后，NAA 80的独特结构特征只留下加工过的肌动蛋白作为NAA 80的体内底物。总之，这项研究揭示了NAA 80 Nt乙酰化的分子和细胞基础，并为开发用于调节细胞骨架特性的抑制剂提供了支架。
N-terminal (Nt) acetylation is a major protein modification catalyzed by N-terminal acetyltransferases (NATs). Methionine acidic N termini, including actin, are cotranslationally Nt acetylated by NatB in all eukaryotes, but animal actins containing acidic N termini, are additionally posttranslationally Nt acetylated by NAA80. Actin Nt acetylation was found to regulate cytoskeletal dynamics and motility, thus making NAA80 a potential target for cell migration regulation. In this work, we developed potent and selective bisubstrate inhibitors for NAA80 and determined the crystal structure of NAA80 in complex with such an inhibitor, revealing that NAA80 adopts a fold similar to other NAT enzymes but with a more open substrate binding region. Furthermore, in contrast to most other NATs, the substrate specificity of NAA80 is mainly derived through interactions between the enzyme and the acidic amino acids at positions 2 and 3 of the actin substrate and not residues 1 and 2. A yeast model revealed that ectopic expression of NAA80 in a strain lacking NatB activity partially restored Nt acetylation of NatB substrates, including yeast actin. Thus, NAA80 holds intrinsic capacity to posttranslationally Nt acetylate NatB-type substrates in vivo. In sum, the presence of a dominant cotranslational NatB in all eukaryotes, the specific posttranslational actin methionine removal in animals, and finally, the unique structural features of NAA80 leave only the processed actins as in vivo substrates of NAA80. Together, this study reveals the molecular and cellular basis of NAA80 Nt acetylation and provides a scaffold for development of inhibitors for the regulation of cytoskeletal properties.