The PKA regulatory subunit from yeast forms a homotetramer: Low-resolution structure of the N-terminal oligomerization domain

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• 作者：Nicolá ; s Gonzá ; lez Bardeci^a ; Julio J. Caramelo^a ; ^b ; Donald K. Blumenthal^c ; Jimena Rinaldi^b ; Silvia Rossi^a ; Silvia Moreno^a ; ^{smoreno@qb.fcen.uba.ar" class="auth_mail" title="E-mail the corresponding author}
• 关键词：AKAPs ; A-Kinase anchoring proteins ; C ; catalytic (subunit) ; CBD ; cAMP binding domain ; CD ; circular dichroism ; D/D domain ; dimerization and docking domain ; Dmax ; maximum intraparticle distance ; EGS ; ethylene glycol bis[succinimidylsuccinate] ; IS ; inhibitory site ; PKA ; protein kinase A ; R ; regulatory (subunit) ; Rg ; radius of gyration ; Rs ; Stokes radius ; s ; sedimentation coefficient ; SAXS ; small-angle X-ray scattering ; SEC ; size-exclusion chromatography ; SLS ; static light scattering
• 刊名：Journal of Structural Biology
• 出版年：2016
• 出版时间：February 2016
• 年：2016
• 卷：193
• 期：2
• 页码：141-154
• 全文大小：1875 K

文摘

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.