Division of Biochemistry, Department of Biology, University of Fribourg Chemin du Musée 10 CH 1700 Fribourg Switzerland.
National School of Tropical Medicine, Baylor College of Medicine Houston TX 77030 USA.
Sci Rep. 2016 Jun 27;6:28838. doi: 10.1038/srep28838.
The production, crystal structure, and functional characterization of the C-terminal cysteine-rich secretory protein/antigen 5/pathogenesis related-1 (CAP) domain of pathogen-related yeast protein-1 (Pry1) from Saccharomyces cerevisiae is presented. The CAP domain of Pry1 (Pry1CAP) is functional in vivo as its expression restores cholesterol export to yeast mutants lacking endogenous Pry1 and Pry2. Recombinant Pry1CAP forms dimers in solution, is sufficient for in vitro cholesterol binding, and has comparable binding properties as full-length Pry1. Two crystal structures of Pry1CAP are reported, one with Mg(2+) coordinated to the conserved CAP tetrad (His208, Glu215, Glu233 and His250) in spacegroup I41 and the other without divalent cations in spacegroup P6122. The latter structure contains four 1,4-dioxane molecules from the crystallization solution, one of which sits in the cholesterol binding site. Both structures reveal that the divalent cation and cholesterol binding sites are connected upon dimerization, providing a structural basis for the observed Mg(2+)-dependent sterol binding by Pry1.
本文介绍了酿酒酵母来源的亲缘酵母蛋白-1（Pry1）的 C 端富含半胱氨酸的分泌蛋白/抗原 5/相关发病机制蛋白-1（CAP）结构域的表达、晶体结构和功能特征。Pry1 的 CAP 结构域（Pry1CAP）在体内具有功能，因为其表达可恢复缺乏内源性 Pry1 和 Pry2 的酵母突变体中的胆固醇外排。重组 Pry1CAP 在溶液中形成二聚体，足以进行体外胆固醇结合，并且具有与全长 Pry1 相当的结合特性。报告了两种 Pry1CAP 的晶体结构，一种结构在 I41 空间群中具有与保守的 CAP 四联体（His208、Glu215、Glu233 和 His250）配位的 Mg2+，另一种结构在 P6122 空间群中没有二价阳离子。后一种结构包含来自结晶溶液的四个 1,4-二恶烷分子，其中一个分子位于胆固醇结合位点。这两种结构都表明，二价阳离子和胆固醇结合位点在二聚化时连接在一起，为观察到的 Pry1 依赖 Mg2+的固醇结合提供了结构基础。
