Department of Biological Chemistry, Facultad de Ciencias Exactas y Naturales, Universidad de Buenos Aires, Ciudad Universitaria, Pabellón 2, 1428 Buenos Aires, Argentina.
Structure. 2010 Nov 10;18(11):1471-82. doi: 10.1016/j.str.2010.08.013.
The major cAMP receptors in eukaryotes are the regulatory (R) subunits of PKA, an allosteric enzyme conserved in fungi through mammals. While mammals have four R-subunit genes, Saccharomyces cerevisiae has only one, Bcy1. To achieve a molecular understanding of PKA activation in yeast and to explore the evolution of cyclic-nucleotide binding (CNB) domains, we solved the structure of cAMP-bound Bcy1(168-416). Surprisingly, the relative orientation of the two CNB domains in Bcy1 is very different from mammalian R-subunits. This quaternary structure is defined primarily by a fungi-specific sequence in the hinge between the αB/αC helices of the CNB-A domain. The unique interface between the two CNB domains in Bcy1 defines the allosteric mechanism for cooperative activation of PKA by cAMP. Some interface motifs are isoform-specific while others, although conserved, play surprisingly different roles in each R-subunit. Phylogenetic analysis shows that structural differences in Bcy1 are shared by fungi of the subphylum Saccharomycotina.
真核生物中主要的 cAMP 受体是 PKA 的调节（R）亚基，PKA 是一种在真菌到哺乳动物中都保守的变构酶。哺乳动物有四个 R 亚基基因，而酿酒酵母（Saccharomyces cerevisiae）只有一个，即 Bcy1。为了从分子水平理解酵母中 PKA 的激活，并探索环核苷酸结合（CNB）结构域的进化，我们解析了 cAMP 结合的 Bcy1（168-416）的结构。令人惊讶的是，Bcy1 中两个 CNB 结构域的相对取向与哺乳动物的 R 亚基非常不同。这种四级结构主要由 CNB-A 结构域的 αB/αC 螺旋之间铰链区中真菌特有的序列决定。Bcy1 中两个 CNB 结构域之间独特的界面定义了 cAMP 协同激活 PKA 的变构机制。一些界面模体是同种型特异性的，而另一些虽然保守，但在每个 R 亚基中却起着截然不同的作用。系统发育分析表明，亚门 Saccharomycotina 的真菌中存在 Bcy1 的结构差异。
