title: Structural and biochemical characterization of human PR70 in isolation and in complex with the scaffolding subunit of protein phosphatase 2A.
abstract: Protein Phosphatase 2A (PP2A) is a major Ser/Thr phosphatase involved in the regulation of various cellular processes. PP2A assembles into diverse trimeric holoenzymes, which consist of a scaffolding (A) subunit, a catalytic (C) subunit and various regulatory (B) subunits. Here we report a 2.0 Å crystal structure of the free B''/PR70 subunit and a SAXS model of an A/PR70 complex. The crystal structure of B''/PR70 reveals a two domain elongated structure with two Ca2+ binding EF-hands. Furthermore, we have characterized the interaction of both binding partner and their calcium dependency using biophysical techniques. Ca2+ biophysical studies with Circular Dichroism showed that the two EF-hands display different affinities to Ca2+. In the absence of the catalytic C-subunit, the scaffolding A-subunit remains highly mobile and flexible even in the presence of the B''/PR70 subunit as judged by SAXS. Isothermal Titration Calorimetry studies and SAXS data support that PR70 and the A-subunit have high affinity to each other. This study provides additional knowledge about the structural basis for the function of B'' containing holoenzymes.
authors.affiliation: Department of Medical Biochemistry and Biophysics, Karolinska Institutet, Stockholm, Sweden.
authors.forename: Rebecca
authors.initials: R
authors.lastname: Dovega
authors.affiliation: Life Science Division, Lawrence Berkeley National Lab (LBNL), Berkeley, California, United States of America.
authors.forename: Susan
authors.initials: S
authors.lastname: Tsutakawa
authors.affiliation: Department of Medical Biochemistry and Biophysics, Karolinska Institutet, Stockholm, Sweden.
authors.forename: Esben M
authors.initials: EM
authors.lastname: Quistgaard
authors.affiliation: Department of Medical Biochemistry and Biophysics, Karolinska Institutet, Stockholm, Sweden; Department of Physics, Chemistry and Biology, Linköping University, Linköping, Sweden.
authors.forename: Madhanagopal
authors.initials: M
authors.lastname: Anandapadamanaban
authors.affiliation: Department of Medical Biochemistry and Biophysics, Karolinska Institutet, Stockholm, Sweden.
authors.forename: Christian
authors.initials: C
authors.lastname: Löw
authors.affiliation: Department of Medical Biochemistry and Biophysics, Karolinska Institutet, Stockholm, Sweden; School of Biological Sciences, Nanyang Technological University, Singapore, Singapore.
authors.forename: Pär
authors.initials: P
authors.lastname: Nordlund
mesh_terms: D001665:Binding Sites; D002118:Calcium / Q000378:metabolism; D002942:Circular Dichroism; D018360:Crystallography, X-Ray; D020035:Holoenzymes / Q000737:chemistry / Q000378:metabolism; D006801:Humans; D008958:Models, Molecular; D055503:Protein Multimerization; D054648:Protein Phosphatase 2 / Q000737:chemistry* / Q000378:metabolism*; D017433:Protein Structure, Secondary; D053838:Scattering, Small Angle; D014961:X-Ray Diffraction / Q000379:methods*
chemical_list: D020035:Holoenzymes; C517191:PPP2R1A protein, human; C432785:PPP2R1B protein, human; C403618:PPP2R3B protein, human; D054648:Protein Phosphatase 2; D002118:Calcium