Gasch Alexander, Wiesner Silke, Martin-Malpartida Pau, Ramirez-Espain Ximena, Ruiz Lidia, Macias Maria J
Structural Biology Program, EMBL Heidelberg, Meyerhofstrasse 1, 69117 Heidelberg, Germany.
J Biol Chem. 2006 Jan 6;281(1):356-64. doi: 10.1074/jbc.M508047200. Epub 2005 Oct 27.
The yeast splicing factor Prp40 (pre-mRNA processing protein 40) consists of a pair of WW domains followed by several FF domains. The region comprising the FF domains has been shown to associate with the 5' end of U1 small nuclear RNA and to interact directly with two proteins, the Clf1 (Crooked neck-like factor 1) and the phosphorylated repeats of the C-terminal domain of RNA polymerase II (CTD-RNAPII). In this work we reported the solution structure of the first FF domain of Prp40 and the identification of a novel ligand-binding site in FF domains. By using chemical shift assays, we found a binding site for the N-terminal crooked neck tetratricopeptide repeat of Clf1 that is distinct and structurally separate from the previously identified CTD-RNAPII binding pocket of the FBP11 (formin-binding protein 11) FF1 domain. No interaction, however, was observed between the Prp40 FF1 domain and three different peptides derived from the CTD-RNAPII protein. Indeed, the equivalent CTD-RNAPII-binding site in the Prp40 FF1 domain is predominantly negatively charged and thus unfavorable for an interaction with phosphorylated peptide sequences. Sequence alignments and phylogenetic tree reconstructions using the FF domains of three functionally related proteins, Prp40, FBP11, and CA150, revealed that Prp40 and FBP11 are not orthologous proteins and supported the different ligand specificities shown by their respective FF1 domains. Our results also revealed that not all FF domains in Prp40 are functionally equivalent. We proposed that at least two different interaction surfaces exist in FF domains that have evolved to recognize distinct binding motifs.
酵母剪接因子Prp40（前体mRNA加工蛋白40）由一对WW结构域和几个FF结构域组成。包含FF结构域的区域已被证明与U1小核RNA的5'端相关联，并直接与两种蛋白质相互作用，即Clf1（弯曲颈样因子1）和RNA聚合酶II C末端结构域的磷酸化重复序列（CTD-RNAPII）。在这项工作中，我们报道了Prp40第一个FF结构域的溶液结构，并鉴定了FF结构域中的一个新的配体结合位点。通过化学位移分析，我们发现了Clf1 N端弯曲颈四肽重复序列的一个结合位点，该位点与先前鉴定的FBP11（formin结合蛋白11）FF1结构域的CTD-RNAPII结合口袋不同且在结构上相互独立。然而，在Prp40 FF1结构域与源自CTD-RNAPII蛋白的三种不同肽之间未观察到相互作用。实际上，Prp40 FF1结构域中的等效CTD-RNAPII结合位点主要带负电荷，因此不利于与磷酸化肽序列相互作用。使用三种功能相关蛋白Prp40、FBP11和CA150 的FF结构域进行序列比对和系统发育树重建，结果表明Prp40和FBP11不是直系同源蛋白，并支持了它们各自FF1结构域所显示的不同配体特异性。我们的结果还表明，Prp40中的并非所有FF结构域在功能上都是等效的。我们提出，FF结构域中至少存在两个不同的相互作用表面，它们已经进化以识别不同的结合基序。
