蛋白质N-末端乙酰化酶NatB的结构和功能

N-terminal acetylation is one of the most common protein modifications in eukaryotes. It is not only involved in regulating protein stability, subcellular localization, and protein-protein interactions, but also plays an important role in cell growth, differentiation, metabolism and tumorigenesis. In eukaryotic cells, six N-terminal acetyltransferases, NatA-F, have been characterized, which belong to the GNAT superfamily. NatB is important in maintaining the structure and function of cytoskeleton, cell proliferation and regulating flowering time and fruit development in plant. NatB complex, composed of a catalytic subunit Naa20 and an auxiliary subunit Naa25, acetylates specifically on those polypeptides with N-terminal Met-Glu-, Met-Asp-, Met-Asn- or Met-Gln-. However, the molecular mechanism of how NatB recognizes and catalyzes these specific substrates is unclear due to lack of its atomic structures. We have successfully expressed and purified the holoenzyme of NatB through a co-expression system in E. coli. We have even determined a crystal structure of NatB in presence of its cofactor CoA in high resolution. In this proposal we will determine a crystal structure in presence of its cognate substrates, which is crucial for us to analyze its unique substrate specificity. We will also devote much of our effort into design and screening of small molecule regulators to the NatB enzyme. This proposal will not only provide a series of crystal structures of NatB complexes, which will help usunderstand the substrate-specific N-terminal acetylation, but also provides a pool of small molecules that have preliminarily demonstrated their potential in NatB inhibition.

N-末端乙酰化是真核细胞中常见的一种蛋白质翻译后修饰方式，可以调节蛋白的稳定性、亚细胞定位和相互作用，更在细胞的生长发育、代谢和癌症发生等过程中起重要的作用。与其它的NAT酶不同，NatB只特异性地乙酰化氨基酸序列上第一位为甲硫氨基酸，第二位为酸性氨基酸及其酰氨等为特点的多肽底物。但它如何识别和催化这些特异性底物的分子机制并不清楚。经过多年的努力，我们已经成功地表达纯化了有活性的NatB全酶，并解析了NatB和辅酶CoA复合体的高分辨率的晶体结构。我们拟继续解析全酶、CoA，并含有其特异性底物的三元复合体的结构，研究NatB识别和催化特异性底物的分子机制，并由此设计筛选一批调节NatB活性的小分子化合物。该研究不仅会解析一系列关键的NatB复合物的结构，从分子水平认识这一类N-末端乙酰化修饰酶的底物特异性，还能提供一个调控NatB活性的前体化合物库，为开发有治疗潜能的小分子抑制剂奠定基础。

N-末端乙酰化是真核细胞中常见的一种蛋白质翻译后修饰方式，可以调节蛋白的稳定性、 亚细胞定位和相互作用，在细胞的生长发育、代谢和癌症发生等过程中起重要作用。NatB特异的乙酰化具有N末端第一位Met，第二位的酸性氨基酸及其酰氨的底物蛋白。我们成功地表达纯化了人、酵母和白色念珠菌的NatB全酶，并解析了白色念珠菌NatB和辅酶CoA 复合体晶体结构。我们也成功地解析了NatB全酶与一个抑制性底物复合体晶体结构， 并利用定点突变和酶活性实验研究了NatB识别和催化特异性底物的关键位点和其反应机制。该结果已发表在Structure（2017）杂志上，但发表在基金资助前几个月，未标注本基金号。我们按研究计划开展了NatB抑制剂的筛选和设计工作。尝试了在NatB结晶过程中结合在活性位点的吗呤乙磺酸（MES），发现没有明显抑制效果。之后，我们利用已经解析的分子结构进行虚拟计算机药物设计。我们首先针对NatB底物结合口袋，从24,982个Drug-like的Zinc小分子库筛选，得到40个top20的小分子。根据小分子与NatB结合方式，挑选14个较好的进行了分子动力学实验，并挑选6个进行50ns的动力学实验，然而，最终得到的5个最优小分子的体外酶活性抑制效果却并不明显。因此，我们又进行了DEL筛选，发现了一个骨架分子acetamido benzamide，目前正在验证过程中。同时，我们解析了一个细菌乙酰化酶VipF和其与底物氯霉素的晶体结构，该工作即将投稿。