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-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.