Evolution of PKA Signaling: Structure of Yeast Regulatory Subunit

RINALDI, J; YANG, J; ROSSI, S; GANAPATHY, S,; MORENO, S.; TAYLOR, SS

New Orleans, Louisiana, USA

Congreso; Experimental Biology 2009-ASBMB; 2009

American Society of Biochemistry and Molecular Biology

cAMP is known to mediate, in both prokaryotes and eukaryotes, a wide variety of cellular responses to external stimuli. In eukaryotes, the major receptor for cAMP signaling is the regulatory subunit (R) of cAMP-dependent protein kinase (PKA). In mammals there are two major classes of R subunits (I and II), however, Saccharomyces cerevisiae has only one R gene (Bcy1). As the yeast is far from mammals in the phylogenetic tree the study of this protein is important from the point of view of molecular evolution. Sequence analysis of the cyclic-AMP binding domain (CNB) has shown that the fungal R subunits belong to a distinct group, yet exhibits some similarity to both mammalian RI and RII. In order to a molecular understanding of the mechanism of activation of PKA by cAMP in yeast and to provide insight into the evolution of cAMP receptor, we purified and crystalized a deletion mutant of Bcy1, Bcy1(168-416) bound to cAMP and then compared it with the previous structures of mammalian RI and RII ? cAMP-bound isoforms. Surprisingly the relative orientation of the two CNB domains in Bcy1 is very different from RI and RIIâ, although each domain superimposes very well. This structure serves as a hallmark for the yeast PKA. Closer examination of the structural differences between mammalian R subunits and Bcy1 identifies a hinge point in the kink between the B and C helices of the A domain that accounts for the major deviation in tertiary structure of the different R subunits. The similarity and differences of the interface between the two CNB domains in Bcy1 was also described and compared with the mammalian isoformsSaccharomyces cerevisiae has only one R gene (Bcy1). As the yeast is far from mammals in the phylogenetic tree the study of this protein is important from the point of view of molecular evolution. Sequence analysis of the cyclic-AMP binding domain (CNB) has shown that the fungal R subunits belong to a distinct group, yet exhibits some similarity to both mammalian RI and RII. In order to a molecular understanding of the mechanism of activation of PKA by cAMP in yeast and to provide insight into the evolution of cAMP receptor, we purified and crystalized a deletion mutant of Bcy1, Bcy1(168-416) bound to cAMP and then compared it with the previous structures of mammalian RI and RII ? cAMP-bound isoforms. Surprisingly the relative orientation of the two CNB domains in Bcy1 is very different from RI and RIIâ, although each domain superimposes very well. This structure serves as a hallmark for the yeast PKA. Closer examination of the structural differences between mammalian R subunits and Bcy1 identifies a hinge point in the kink between the B and C helices of the A domain that accounts for the major deviation in tertiary structure of the different R subunits. The similarity and differences of the interface between the two CNB domains in Bcy1 was also described and compared with the mammalian isoforms