Tetrameric structure and interactors of the N-terminus of Bcy1, the regulatory subunit of PKA from yeast

TOFOLÓN E; GONZÁLEZ BARDECI N; VALACCO P; FERNÁNDEZ G; NEME TAUIL R; ROSSI S; MORENO S

Mar del Plata

Congreso; LI reunión de SAIB; 2015

Sociedad Argentina de Investigación en Bioquímica y Biología Molecular

PK A is classically a tetramer formed by a dimer of regulatory subunit (R2), which binds cAMP, and two catalytic subunits. In mammals the N-terminus of R2 (DD) is responsible for dimerization and for binding to AKAPs, through an hydrophobic surface. We have shown that Bcy1, the yeast PKA R subunit: 1) binds to specific interactors depending on its N-term 85 aa; 2) the interaction is dependent on charged residues; 3) a bacterial recombinant construct of Bcy1 (1-50) is a tetramer (dimer of dimers) both in crystal and solution. We now present characteristics of the tetrameric structure showing the importance of the orientation of Arg45 in the maintenance of the tetramer, in comparison with mammalian structures/sequences in a complete phylogenetic analysis. Two mutants that could affect the tetramerization are constructed and purified: Arg45Ala, and deltaQ43. In order to study whether Bcy1 (1-50) is sufficient to interact with specific proteins we overexpressed Tag.Bcy1(1-50) in a WT yeast strain; interactors were pulled-down with Ni-agarose; eluted with imidazol and analyzed by nanoHPLC-ESI-Orbitrap MSMS and compared to a control WT strain. Among the specific binders was endogenous Bcy1. This result indicates that Tag-Bcy1(1-50) and endogenous Bcy1 interact in vivo and raises the doubt on whether the specific interactors bind to the Tag-.DD domain directly or via endogenous Bcy1.