Kurimoto K, Fukai S, Nureki O, Muto Y, Yokoyama S
Department of Biophysics and Biochemistry, Graduate School of Science, The University of Tokyo, 7-3-1 Hongo, Bunkyo-Ku, Tokyo 113-0033, Japan.
Structure. 2001 Dec;9(12):1253-63. doi: 10.1016/s0969-2126(01)00686-4.
The AU binding homolog of enoyl-CoA hydratase (AUH) is a bifunctional protein that has two distinct activities: AUH binds to RNA and weakly catalyzes the hydration of 2-trans-enoyl-coenzyme A (enoyl-CoA). AUH has no sequence similarity with other known RNA binding proteins, but it has considerable sequence similarity with enoyl-CoA hydratase. A segment of AUH, named the R peptide, binds to RNA. However, the mechanism of the RNA binding activity of AUH remains to be elucidated.
We determined the crystal structure of human AUH at 2.2 A resolution. AUH adopts the typical fold of the enoyl-CoA hydratase/isomerase superfamily and forms a hexamer as a dimer of trimers. Interestingly, the surface of the AUH hexamer is positively charged, in striking contrast to the negatively charged surfaces of the other members of the superfamily. Furthermore, wide clefts are uniquely formed between the two trimers of AUH and are highly positively charged with the Lys residues in alpha helix H1, which is located on the edge of the cleft and contains the majority of the R peptide. A mutational analysis showed that the lysine residues in alpha helix H1 are essential to the RNA binding activity of AUH.
Alpha helix H1 exposes a row of Lys residues on the solvent-accessible surface. These characteristic Lys residues are named the "lysine comb." The distances between these Lys residues are similar to those between the RNA phosphate groups, suggesting that the lysine comb may continuously bind to a single-stranded RNA. The clefts between the trimers may provide spaces sufficient to accommodate the RNA bases.
烯酰辅酶A水合酶（AUH）的AU结合同源物是一种具有两种不同活性的双功能蛋白：AUH与RNA结合并弱催化2-反式烯酰辅酶A（烯酰辅酶A）的水合作用。AUH与其他已知的RNA结合蛋白没有序列相似性，但与烯酰辅酶A水合酶有相当大的序列相似性。AUH的一段名为R肽的片段与RNA结合。然而，AUH的RNA结合活性机制仍有待阐明。
我们以2.2埃的分辨率确定了人AUH的晶体结构。AUH采用烯酰辅酶A水合酶/异构酶超家族的典型折叠结构，形成三聚体二聚体的六聚体。有趣的是，AUH六聚体的表面带正电荷，这与超家族其他成员带负电荷的表面形成鲜明对比。此外，在AUH的两个三聚体之间独特地形成了宽裂缝，并且位于裂缝边缘且包含大部分R肽的α螺旋H1中的赖氨酸残基使其高度带正电荷。突变分析表明，α螺旋H1中的赖氨酸残基对AUH的RNA结合活性至关重要。
α螺旋H1在溶剂可及表面暴露了一排赖氨酸残基。这些具有特征性的赖氨酸残基被称为“赖氨酸梳”。这些赖氨酸残基之间的距离与RNA磷酸基团之间的距离相似，这表明赖氨酸梳可能连续结合单链RNA。三聚体之间的裂缝可能提供足以容纳RNA碱基的空间。
