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J Struct Biol ; 193(2): 141-54, 2016 Feb.
Article in English | MEDLINE | ID: mdl-26687415

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.


Subject(s)
Cyclic AMP-Dependent Protein Kinases/chemistry , Saccharomyces cerevisiae Proteins/chemistry , Chromatography, Gel , Circular Dichroism , Cyclic AMP-Dependent Protein Kinases/metabolism , Models, Molecular , Protein Multimerization , Protein Structure, Tertiary , Protein Subunits/chemistry , Saccharomyces cerevisiae Proteins/genetics , Saccharomyces cerevisiae Proteins/metabolism , Scattering, Small Angle , X-Ray Diffraction
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