Maguire Mahon L, Guler-Gane Gulin, Nietlispach Daniel, Raine Andrew R C, Zorn Aaron M, Standart Nancy, Broadhurst R William
Department of Biochemistry, University of Cambridge, 80 Tennis Court Road, Cambridge CB2 1GA, UK.
J Mol Biol. 2005 Apr 29;348(2):265-79. doi: 10.1016/j.jmb.2005.02.058.
The Quaking protein belongs to the family of STAR/GSG domain RNA-binding proteins and is involved in multiple cell signalling and developmental processes in vertebrates, including the formation of myelin. Heteronuclear NMR methods were used to determine the solution structure of a 134 residue fragment spanning the KH and QUA2 homology regions of the Quaking protein from Xenopus laevis (pXqua) in the absence of RNA. The protein is shown to adopt an extended type I KH domain fold that is connected to a structured alpha-helix in the C-terminal QUA2 region by means of a highly flexible linker. A comparison with the solution structure of the related protein splicing factor 1 (SF1) indicates that most aspects of the RNA-binding interface are conserved in pXqua, although the "variable loop" region that follows the second beta-strand possesses two additional alpha-helices. The structure of pXqua provides an appropriate template for building models of important homologues, such as GLD-1 and Sam68. Measurements of the (15)N relaxation parameters of pXqua confirm that the polypeptide backbone of the QUA2 region is more dynamic than that of the KH portion, and that the C-terminal helix is partially structured in the absence of RNA. By comparison with a random coil reference state, the nascent structure in the QUA2 region is estimated to contribute 15.5kJmol(-1) to the change in conformational free energy that occurs on forming a complex with RNA. Since STAR/GSG proteins may regulate alternative splicing by competing with SF1 in the nucleus for specific branch-point sequences that signal intronic RNA, the formation of secondary structure in the QUA2 region in the unbound state of pXqua has important functional consequences.
震颤蛋白属于STAR/GSG结构域RNA结合蛋白家族，参与脊椎动物的多种细胞信号传导和发育过程，包括髓鞘形成。在没有RNA的情况下，采用异核核磁共振方法测定了非洲爪蟾（pXqua）震颤蛋白跨越KH和QUA2同源区域的134个残基片段的溶液结构。结果表明，该蛋白呈现出一种延伸型I KH结构域折叠，通过高度灵活的连接子与C端QUA2区域的结构化α螺旋相连。与相关蛋白剪接因子1（SF1）的溶液结构比较表明，尽管在第二条β链之后的“可变环”区域有两个额外的α螺旋，但pXqua中RNA结合界面的大多数方面是保守的。pXqua的结构为构建重要同源物（如GLD-1和Sam68）的模型提供了合适的模板。对pXqua的（15）N弛豫参数的测量证实，QUA2区域的多肽主链比KH部分更具动态性，并且在没有RNA的情况下C端螺旋部分结构化。与随机卷曲参考态相比，估计QUA2区域的新生结构对与RNA形成复合物时发生的构象自由能变化贡献15.5kJmol（-1）。由于STAR/GSG蛋白可能通过在细胞核中与SF1竞争信号内含子RNA的特定分支点序列来调节可变剪接，因此pXqua未结合状态下QUA2区域二级结构的形成具有重要的功能后果。
