title: The PKA regulatory subunit from yeast forms a homotetramer: Low-resolution structure of the N-terminal oligomerization domain.
abstract: The cAMP dependent protein kinase (PKA) is a key enzyme involved in many cellular processes in eukaryotes. In mammals, the regulatory (R) subunit localises the catalytic (C) subunit to specific subcellular sites through the interaction of its N-terminal homodimeric docking and dimerization (D/D) domain with specific scaffold proteins. The structure of the D/D domain has been extensively studied in mammals, but there is little information from non-mammalian species. In this work, we present the structural analysis of the D/D domain of Bcy1, the R subunit of PKA from Saccharomyces cerevisiae. Using chemical crosslinking experiments and static light scattering measurements we found that this R subunit forms a tetramer in solution, unlike its dimeric mammalian counterparts. We determined that the D/D domain is responsible for this unusual oligomeric state. Using biophysical techniques including size-exclusion chromatography, sucrose gradient sedimentation, small angle X-ray scattering (SAXS), and circular dichroism, we performed a detailed structural characterization of the tetrameric D/D domain of Bcy1. We used homology modelling in combination with computer-aided docking methods and ab initio SAXS modelling methods to develop structural models for the D/D domain tetramer. The models consist of two homodimers with a canonical D/D domain fold that generate a dimer of dimers with novel putative interaction surfaces. These findings indicate that the oligomerization states of PKA R subunits is more diverse than previously thought, and suggest that this might allow some forms of PKA to interact with a wide range of intracellular partners.
authors.affiliation: Departamento de Química Biológica, IQUIBICEN-CONICET, Facultad de Ciencias Exactas y Naturales, Universidad de Buenos Aires, C1428EGA Buenos Aires, Argentina.
authors.forename: Nicolás
authors.initials: N
authors.lastname: González Bardeci
authors.affiliation: Departamento de Química Biológica, IQUIBICEN-CONICET, Facultad de Ciencias Exactas y Naturales, Universidad de Buenos Aires, C1428EGA Buenos Aires, Argentina; Fundación Instituto Leloir, IIBBA-CONICET, C1405BWE Buenos Aires, Argentina.
authors.forename: Julio J
authors.initials: JJ
authors.lastname: Caramelo
authors.affiliation: Department of Pharmacology & Toxicology, University of Utah, Salt Lake City, UT 84112, USA.
authors.forename: Donald K
authors.initials: DK
authors.lastname: Blumenthal
authors.affiliation: Fundación Instituto Leloir, IIBBA-CONICET, C1405BWE Buenos Aires, Argentina.
authors.forename: Jimena
authors.initials: J
authors.lastname: Rinaldi
authors.affiliation: Departamento de Química Biológica, IQUIBICEN-CONICET, Facultad de Ciencias Exactas y Naturales, Universidad de Buenos Aires, C1428EGA Buenos Aires, Argentina.
authors.forename: Silvia
authors.initials: S
authors.lastname: Rossi
authors.affiliation: Departamento de Química Biológica, IQUIBICEN-CONICET, Facultad de Ciencias Exactas y Naturales, Universidad de Buenos Aires, C1428EGA Buenos Aires, Argentina. Electronic address: smoreno@qb.fcen.uba.ar.
authors.forename: Silvia
authors.initials: S
authors.lastname: Moreno
mesh_terms: D002850:Chromatography, Gel; D002942:Circular Dichroism; D017868:Cyclic AMP-Dependent Protein Kinases / Q000737:chemistry* / Q000378:metabolism; D008958:Models, Molecular; D055503:Protein Multimerization; D017434:Protein Structure, Tertiary; D021122:Protein Subunits / Q000737:chemistry; D029701:Saccharomyces cerevisiae Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D053838:Scattering, Small Angle; D014961:X-Ray Diffraction
chemical_list: C574570:Bcy1 protein, S cerevisiae; D021122:Protein Subunits; D029701:Saccharomyces cerevisiae Proteins; D017868:Cyclic AMP-Dependent Protein Kinases
keywords: Bcy1; D/D domain; Protein kinase A; Regulatory subunit; Saccharomyces cerevisiae