Díaz Casas Adalberto, Chazin Walter J, Pastrana-Ríos Belinda
Department of Chemistry, University of Puerto Rico Mayagüez Campus, Mayagüez, Puerto Rico.
Department of Biochemistry and Chemistry, Center for Structural Biology, Vanderbilt University, Nashville, Tennessee.
Biophys J. 2017 Jun 20;112(12):2529-2539. doi: 10.1016/j.bpj.2017.03.042.
Pre-mRNA processing protein 40 (Prp40) is a nuclear protein that has a role in pre-mRNA splicing. Prp40 possesses two leucine-rich nuclear export signals, but little is known about the function of Prp40 in the export process. Another protein that has a role in protein export is centrin, a member of the EF-hand superfamily of Ca-binding proteins. Prp40 was found to be a centrin target by yeast-two-hybrid screening using both Homo sapiens centrin 2 (Hscen2) and Chlamydomonas reinhardtii centrin (Crcen). We identified a centrin-binding site within H. sapiens Prp40 homolog A (HsPrp40A), which contains a hydrophobic triad WLL that is known to be important in the interaction with centrin. This centrin-binding site is highly conserved within the first nuclear export signal consensus sequence identified in Saccharomyces cerevisiae Prp40. Here, we examine the interaction of HsPrp40A peptide (HsPrp40Ap) with both Hscen2 and Crcen by isothermal titration calorimetry. We employed the thermodynamic parameterization to estimate the polar and apolar surface area of the interface. In addition, we have defined the molecular mechanism of thermally induced unfolding and dissociation of the Crcen-HsPrp40Ap complex using two-dimensional infrared correlation spectroscopy. These complementary techniques showed for the first time, to our knowledge, that HsPrp40Ap interacts with centrin in vitro, supporting a coupled functional role for these proteins in pre-mRNA splicing.
前体mRNA加工蛋白40（Prp40）是一种在细胞核中发挥作用的蛋白质，参与前体mRNA剪接过程。Prp40具有两个富含亮氨酸的核输出信号，但对于Prp40在输出过程中的功能了解甚少。另一种在蛋白质输出中发挥作用的蛋白质是中心蛋白，它是钙结合蛋白EF-手超家族的成员。通过使用人类中心蛋白2（Hscen2）和莱茵衣藻中心蛋白（Crcen）进行酵母双杂交筛选，发现Prp40是中心蛋白的靶点。我们在人类Prp40同源物A（HsPrp40A）中鉴定出一个中心蛋白结合位点，该位点包含一个疏水三联体WLL，已知其在与中心蛋白的相互作用中很重要。这个中心蛋白结合位点在酿酒酵母Prp40中鉴定出的第一个核输出信号共有序列内高度保守。在此，我们通过等温滴定量热法研究了HsPrp40A肽（HsPrp40Ap）与Hscen2和Crcen的相互作用。我们采用热力学参数化来估计界面的极性和非极性表面积。此外，我们使用二维红外光谱相关技术定义了Crcen-HsPrp40Ap复合物热诱导解折叠和解离的分子机制。据我们所知，这些互补技术首次表明HsPrp40Ap在体外与中心蛋白相互作用，支持了这些蛋白质在前体mRNA剪接中具有耦合功能作用。
