Structural basis of cofactor-mediated stabilization and substrate recognition of the α-tubulin acetyltransferase αTAT1
Structural basis of cofactor-mediated stabilization and substrate recognition of the α-tubulin acetyltransferase αTAT1
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α-微管蛋白乙酰转移酶 αTAT1 辅因子介导的稳定和底物识别的结构基础
DOI：
10.1042/bj20141193
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发表时间：
2015
影响因子：
4.1
通讯作者：
Junya Hayase and Hideki Sumimoto
中科院分区：
文献类型：
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作者：
Satoru Yuzawa;Sachiko Kamakura;Junya Hayase and Hideki Sumimoto
微管的功能部分受微管蛋白翻译后修饰的控制，包括α-微管蛋白中Lys 40的乙酰化修饰。α-微管蛋白乙酰基转移酶1（aTAT 1）是真核生物中进化上保守的一种酶，最近被鉴定为α-微管蛋白Lys 40乙酰基转移酶，其中乙酰辅酶A（acetyl-CoA）作为乙酰基供体。然而，这种酶的调节和底物识别尚未完全了解。在本研究中，我们表明，乙酰辅酶A和辅酶A各自形成一个稳定的复合物与人的aTAT 1，以保持蛋白质的完整性，在体内和体外。aTAT 1中的不变残基Arg 132和Ser 160不仅参与与AcCoA的稳定相互作用，而且还参与与CoA的稳定相互作用，这一点得到了与CoA复合物中α TAT 1催化结构域当前晶体结构分析的支持。在缺乏CoA和AcCoA的情况下，丙氨酸取代Arg 132或Ser 160会导致分离的α TAT 1催化结构域发生剧烈的错误折叠，但在存在过量辅因子的情况下则不会。当在哺乳动物Madin-Darby犬肾细胞中表达时，携带R132 A或S160 A取代的突变体α TAT 1比野生型蛋白降解快得多。此外，用含Lys 40的肽进行的丙氨酸扫描实验表明，α-微管蛋白Ser 38对α TAT 1的底物识别至关重要，而Asp 39、Ile 42、甘氨酸段（氨基酸残基43-45）和Asp 46也参与其中。对底物选择的要求与各种组蛋白乙酰转移酶完全不同，这似乎与α TAT 1不能乙酰化组蛋白一致。
The functions of microtubules are controlled in part by tubulin post-translational modification including acetylation of Lys40in α-tubulin. aTAT1 (α-tubulin acetyltransferase 1), an enzyme evolutionarily conserved among eukaryotes, has recently been identified as the major α-tubulin Lys40acetyltransferase, in which AcCoA (acetyl-CoA) serves as an acetyl group donor. The regulation and substrate recognition of this enzyme, however, have not been fully understood. In the present study, we show that AcCoA and CoA each form a stable complex with human aTAT1 to maintain the protein integrity both in vivo and in vitro. The invariant residues Arg132and Ser160in aTAT1 participate in the stable interaction not only with AcCoA but also with CoA, which is supported by analysis of the present crystal structures of the αTAT1 catalytic domain in complex with CoA. Alanine substitution for Arg132or Ser160leads to a drastic misfolding of the isolated αTAT1 catalytic domain in the absence of CoA and AcCoA but not in the presence of excess amounts of either cofactor. A mutant αTAT1 carrying the R132A or S160A substitution is degraded much faster than the wild-type protein when expressed in mammalian Madin-Darby canine kidney cells. Furthermore, alanine-scanning experiments using Lys40-containing peptides reveal that α-tubulin Ser38is crucial for substrate recognition of αTAT1, whereas Asp39, Ile42, the glycine stretch (amino acidresidues 43-45) and Asp46are also involved. The requirement for substrate selection is totally different from that in various histone acetyltransferases, which appears to be consistent with the inability of αTAT1 to acetylate histones.