Solution structure of Bcy1, the regulatory subunit of yeast protein kinase A

LUCAS FERNáNDEZ NúñEZ; SILVIA ROSSI; DONALD BLUMENTHAL; SILVIA MORENO

Fos de Iguazu

Congreso; XXXVII Reunión Anual de la Sociedad Brasileña de Bioquímica y Biología Molecular; 2010

SSBq

The major receptor for cAMP signaling in eukaryotes is the regulatory subunit (R) of protein kinase A. In mammals there are two classes of R subunits (I and II); however, Saccharomyces cerevisiae has only one R(Bcy1). R subunits are dimers comprised by a dimerization/docking domain in the N terminus, a hinge region, and two tanden cAMP binding domains (CNB) at the C-terminus. The crystal structure of Bcy1(168-416) has been solved. The hallmark of this structure is the relative orientation of the two CNB domains, different from RIá and RIIâ. We now present the study of the solution structure of Bcy1 by SAXS. We have measured the scattering of the whole molecule, of a dimer with a deletion of CNB B, and of a monomer D140 Bcy1. The Rg (gyration radius) and dmax (derived from the P(r) function) of the molecules were calculated.The structure of D140 Bcy1 was modelled from ab initio using the DAMMIN program of the ATSAS package and the predicted structure was compared with the crystal structure. The results indicate that solution and crystal structures of the cAMP binding domains are alike, confirming the hallmark difference of Bcy1 in this region. The structure of the whole dimer is predicted to be much more compact than Ri and RII. Free R subuntis are unstable molecules, and easily break apart. In order to have more information for the future modelling ot the whole dimer, using the SAXS data, an analysis of the labile regions in Bcy1 was determined by MALDI-TOF-TOF analysis.