CRYSTAL STRUCTURE OF THE REGULATORY SUBUNIT

RINALDI , J., WU, J.,YANG, J., ROSSI, S., MORENO, S., TAYLOR, S

Córdoba, Carlos Paz

Congreso; XLIV Reunión Anual SAIB; 2008

Sociedad Argentina de investigaciones Bioquímicas

SB-C03. CRYSTAL STRUCTURE OF THE REGULATORY SUBUNIT OF PKA FROM Saccharomyces cerevisiae Rinaldi J , Wu J , Yang J , Rossi S , Moreno S , Taylor SS Departamento de Química Biológica, FCEyN-UBA. Chemistry & Biochemistry Department, UCSD.CA, USA. E-mail: jrinaldi@qb.fcen.uba.ar In mammals, the PKA holoenzyme exists as a complex of two catalytic subunits and a regulatory (R) subunit dimer. R subunits have a dimerization and docking domain at the N terminus; at the C terminus, two tandem cAMP-binding domains and in between a flexible hinge region, including a substrate-like inhibitor sequence that docks to the active site cleft of the C subunit. Much effort has been put in studying structure-function relationships in mammalian PKAs. The aim of this work is to solve the structure of the R subunit from Saccharomyces cerevisiae, one of the most intensively studied eukaryotic model organism in signal transduction. Unlike mammals, that have been shown to have two major classes of R subunits (I and II), S. cerevisiae has only one R gene. We overexpressed and purified different deletion mutants of the protein. The mutant lacking the first 167 residues was crystallized. We report the crystal structure of the protein bound to cAMP, which we determined to 2.20 Å resolution and refined to an R factor of 0,200 with an R free factor of 0,263. The new structure shows two tandem cAMP binding domains, which relative orientation is different from the one of mammalian R subunits. The two domains are more tightly bound to each other. The R subunit from S.cerevisiae represent a new type of R subunit, as it has different features from RI and RII.