LOW R E SOLUT ION S TRUCTURE OF THE DIMERIZATION DOMAIN OF BCY1, THE REGULATORY SUBUNITOFYEASTPKA

GONZÁLEZ BARDECI, N; CARAMELO, J; BLUMENTHAL, D; FERNÁNDEZ NÚÑES, L; TURJANSKI, A; ROSSI, S; MORENO, S

Mendoza

Congreso; XLVIII Reunión Anual de la Sociedad Argentina de Bioquímica y Biología Molecular (SAIB); 2012

SAIB

The regulatory (R) subunit of protein kinase A (PKA) from mammals is dimeric in solution. TheNtermini from both protomers constitute the docking and dimerization (DD) domain, responsible for the quaternary structure and for the subcellular localization of the molecule. This domain consists of an X type four helix bundle fold. No structural study of this domain from other organisms is available. Here we present the first structural characterization of the N terminus of Bcy1, the R subunit of PKA from Saccharomyces cerevisiae. Using multiple sequence alignments, secondary structure prediction, and chemical crosslinking, we have shown that Bcy1 is dimeric in solution and have mapped the region of the molecule responsible for dimerization. The first 50 aminoacids of Bcy1 were cloned and overexpressed in E.coli. This purified fragment was shown to be dimeric both by chemical crosslinking and gel filtration, providing evidence that this is the dimerization domain of Bcy1. Circular dichroism shows that it is highly helical, thus resembling DD from mammals. Its melting temperature, followed by ellipticity at 221 nm, is concentration dependent, supporting its oligomeric nature. Denaturation is highly reversible, suggesting a great stability. SAXS experiments were also performed on this domain. These results suggest that the structure of this domain is well conserved through evolution.