Structural insights into mechanism and specificity of O-GlcNAc transferase.
Structural insights into mechanism and specificity of O-GlcNAc transferase.
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DOI：
10.1038/emboj.2008.186
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发表时间：
2008-10-22
期刊：
影响因子：
11.4
通讯作者：
van Aalten, Daan M. F.
中科院分区：
文献类型：
--
作者：
Clarke, Andrew J.;Hurtado-Guerrero, Ramon;Pathak, Shalini;Schuttelkopf, Alexander W.;Borodkin, Vladimir;Shepherd, Sharon M.;Ibrahim, Adel F. M.;van Aalten, Daan M. F.
N-乙酰氨基葡萄糖(O-GlcNAc)对蛋白质丝氨酸/苏氨酸的翻译后修饰是动态的、可诱导的和丰富的，通过干扰蛋白质磷酸化来调节许多细胞过程。O-GlcN酰化受O-GlcNAc转移酶(OGT)和O-GlcNAcase调控，这两种酶都是由后生动物基因组中的单个必需基因编码的。目前还不清楚OGT如何识别其糖核苷酸供体并将O-GlcNAc转移到蛋白质/肽上，以及该酶如何识别特定的细胞蛋白质底物。在这里，我们通过X射线结晶学和诱变实验表明，OGT采用了(金属无关)GT-B折叠，并在一个高度保守的推定的肽结合槽的底部结合了UDP-GlcNAc类似物，该槽被一个移动环覆盖。值得注意的是，四肽重复序列(TPR)与活性部位紧密相互作用，形成一个连续的120°相互作用面，而先前预测的磷脂酰肌醇结合部位位于催化结构域的另一端。在结构的基础上，我们确定了TPR的截断/点突变，这些突变体对蛋白质/肽的活性具有不同的影响，从而首次深入了解OGT可能如何识别其底物。
Post-translational modification of protein serines/threonines with N-acetylglucosamine (O-GlcNAc) is dynamic, inducible and abundant, regulating many cellular processes by interfering with protein phosphorylation. O-GlcNAcylation is regulated by O-GlcNAc transferase (OGT) and O-GlcNAcase, both encoded by single, essential, genes in metazoan genomes. It is not understood how OGT recognises its sugar nucleotide donor and performs O-GlcNAc transfer onto proteins/peptides, and how the enzyme recognises specific cellular protein substrates. Here, we show, by X-ray crystallography and mutagenesis, that OGT adopts the (metal-independent) GT-B fold and binds a UDP-GlcNAc analogue at the bottom of a highly conserved putative peptide-binding groove, covered by a mobile loop. Strikingly, the tetratricopeptide repeats (TPRs) tightly interact with the active site to form a continuous 120 Å putative interaction surface, whereas the previously predicted phosphatidylinositide-binding site locates to the opposite end of the catalytic domain. On the basis of the structure, we identify truncation/point mutants of the TPRs that have differential effects on activity towards proteins/peptides, giving first insights into how OGT may recognise its substrates.
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