Barlow P N, Luisi B, Milner A, Elliott M, Everett R
Department of Biochemistry, Oxford University, England, U.K.
J Mol Biol. 1994 Mar 25;237(2):201-11. doi: 10.1006/jmbi.1994.1222.
A recently identified sequence motif, referred to as "C3HC4" (also "RING finger" and "A Box") for its distinctive pattern of putative metal-binding residues, has been found in a wide range of proteins. In a previous paper we described the expression and purification of fragments encompassing this motif from the Vmw110 (IPC0) protein family. We showed that the equine herpes virus protein binds zinc ions and adopts a beta beta alpha beta fold. We now report the tertiary structure of this domain in solution, as determined by two-dimensional 1H-NMR An amphipathic alpha-helix lies along one surface of a triple-stranded beta-sheet. Four pairs of metal-binding residues sequester two zincs at distinct tetrahedral sites. The first and third pairs bind one metal ion, while the second and fourth pairs bind the other, forming an interleaved whole. The first and the fourth pairs are contained within two prominent, well-defined loops related by an approximate dyad symmetry. Conserved residues within the helix, sheet and loops contribute to a compact hydrophobic core. The region comprising the first two beta-strands and the alpha-helix has remarkable structural similarity with a TFIIIA type of zinc finger, even though the C3HC4 domain appears not to bind specifically to DNA or RNA. Using site-directed mutagenesis we demonstrate that exposed polar side-chains of the C3HC4 alpha-helix are essential for trans-activation of gene expression by an intact herpes virus regulatory protein.
最近发现了一种序列基序，因其假定的金属结合残基的独特模式而被称为“C3HC4”（也称为“环指”和“A框”），在多种蛋白质中都有发现。在之前的一篇论文中，我们描述了从Vmw110（IPC0）蛋白家族中包含该基序的片段的表达和纯化。我们表明，马疱疹病毒蛋白结合锌离子并采用ββαβ折叠结构。我们现在报告通过二维1H-NMR测定的该结构域在溶液中的三级结构。一个两亲性α螺旋沿着三链β折叠的一个表面排列。四对金属结合残基在不同的四面体位置螯合两个锌离子。第一对和第三对结合一个金属离子，而第二对和第四对结合另一个金属离子，形成一个交错的整体。第一对和第四对包含在两个由近似二分对称相关的突出、明确的环内。螺旋、折叠和环内的保守残基形成一个紧密的疏水核心。尽管C3HC4结构域似乎不与DNA或RNA特异性结合，但包含前两个β链和α螺旋的区域与TFIIIA型锌指具有显著的结构相似性。通过定点诱变，我们证明C3HC4α螺旋暴露的极性侧链对于完整的疱疹病毒调节蛋白对基因表达的反式激活至关重要。
