Han Sang Hee, Ha Jun Yong, Kim Kyoung Hoon, Oh Sung Jin, Kim Do Jin, Kang Ji Yong, Yoon Hye Jin, Kim Se-Hee, Seo Ji Hae, Kim Kyu-Won, Suh Se Won
Department of Chemistry, College of Natural Sciences, Seoul National University, Seoul 151-742, South Korea.
Acta Crystallogr Sect F Struct Biol Cryst Commun. 2006 Nov 1;62(Pt 11):1127-30. doi: 10.1107/S1744309106040267. Epub 2006 Oct 20.
N-terminal acetylation is one of the most common protein modifications in eukaryotes, occurring in approximately 80-90% of cytosolic mammalian proteins and about 50% of yeast proteins. ARD1 (arrest-defective protein 1), together with NAT1 (N-acetyltransferase protein 1) and possibly NAT5, is responsible for the NatA activity in Saccharomyces cerevisiae. In mammals, ARD1 is involved in cell proliferation, neuronal development and cancer. Interestingly, it has been reported that mouse ARD1 (mARD1(225)) mediates epsilon-acetylation of hypoxia-inducible factor 1alpha (HIF-1alpha) and thereby enhances HIF-1alpha ubiquitination and degradation. Here, the preliminary X-ray crystallographic analyses of two N-terminal acetyltransferase-related proteins encoded by the Ta0058 and Ta1140 genes of Thermoplasma acidophilum are reported. The Ta0058 protein is related to an N-terminal acetyltransferase complex ARD1 subunit, while Ta1140 is a putative N-terminal acetyltransferase-related protein. Ta0058 shows 26% amino-acid sequence identity to both mARD1(225) and human ARD1(235). The sequence identity between Ta0058 and Ta1140 is 28%. Ta0058 and Ta1140 were overexpressed in Escherichia coli fused with an N-terminal purification tag. Ta0058 was crystallized at 297 K using a reservoir solution consisting of 0.1 M sodium acetate pH 4.6, 8%(w/v) polyethylene glycol 4000 and 35%(v/v) glycerol. X-ray diffraction data were collected to 2.17 A. The Ta0058 crystals belong to space group P4(1) (or P4(3)), with unit-cell parameters a = b = 49.334, c = 70.384 A, alpha = beta = gamma = 90 degrees. The asymmetric unit contains a monomer, giving a calculated crystal volume per protein weight (V(M)) of 2.13 A(3) Da(-1) and a solvent content of 42.1%. Ta1140 was also crystallized at 297 K using a reservoir solution consisting of 0.1 M trisodium citrate pH 5.6, 20%(v/v) 2-propanol, 20%(w/v) polyethylene glycol 4000 and 0.2 M sodium chloride. X-ray diffraction data were collected to 2.40 A. The Ta1140 crystals belong to space group R3, with hexagonal unit-cell parameters a = b = 75.174, c = 179.607 A, alpha = beta = 90, gamma = 120 degrees. Two monomers are likely to be present in the asymmetric unit, with a V(M) of 2.51 A(3) Da(-1) and a solvent content of 51.0%.
N 端乙酰化是真核生物中最常见的蛋白质修饰之一，约80% - 90%的胞质哺乳动物蛋白和约50%的酵母蛋白会发生这种修饰。ARD1（arrest - defective protein 1，阻滞缺陷蛋白1）与NAT1（N - 乙酰转移酶蛋白1）以及可能的NAT5一起，负责酿酒酵母中的NatA活性。在哺乳动物中，ARD1参与细胞增殖、神经元发育和癌症。有趣的是，据报道小鼠ARD1（mARD1(225)）介导缺氧诱导因子1α（HIF - 1α）的ε - 乙酰化，从而增强HIF - 1α的泛素化和降解。在此，报道了嗜热栖热菌Ta0058和Ta1140基因编码的两种N端乙酰转移酶相关蛋白的初步X射线晶体学分析。Ta0058蛋白与N端乙酰转移酶复合物ARD1亚基相关，而Ta1140是一种推定的N端乙酰转移酶相关蛋白。Ta0058与mARD1(225)和人ARD1(235)的氨基酸序列同一性均为26%。Ta0058和Ta1140之间的序列同一性为28%。Ta0058和Ta1140与N端纯化标签融合后在大肠杆菌中过表达。Ta0058在297 K下使用由0.1 M乙酸钠pH 4.6、8%（w/v）聚乙二醇4000和35%（v/v）甘油组成的储液进行结晶。X射线衍射数据收集到2.17 Å。Ta0058晶体属于空间群P4(1)（或P4(3)），晶胞参数a = b = 49.334，c = 70.384 Å，α = β = γ = 90°。不对称单元包含一个单体，计算得出的每蛋白重量晶体体积（V(M)）为2.13 ų Da⁻¹，溶剂含量为42.1%。Ta1140也在297 K下使用由0.1 M柠檬酸三钠pH 5.6、20%（v/v）异丙醇、20%（w/v）聚乙二醇4000和0.2 M氯化钠组成的储液进行结晶。X射线衍射数据收集到2.40 Å。Ta1140晶体属于空间群R3，六方晶胞参数a = b = 75.174，c = 179.607 Å，α = β = 9
