title: Realizing the allosteric potential of the tetrameric protein kinase A RIα holoenzyme.
abstract: PKA holoenzymes containing two catalytic (C) subunits and a regulatory (R) subunit dimer are activated cooperatively by cAMP. While cooperativity involves the two tandem cAMP binding domains in each R-subunit, additional cooperativity is associated with the tetramer. Of critical importance is the flexible linker in R that contains an inhibitor site (IS). While the IS becomes ordered in the R:C heterodimer, the overall conformation of the tetramer is mediated largely by the N-Linker that connects the D/D domain to the IS. To understand how the N-Linker contributes to assembly of tetrameric holoenzymes, we engineered a monomeric RIα that contains most of the N-Linker, RIα(73-244), and crystallized a holoenzyme complex. Part of the N-linker is now ordered by interactions with a symmetry-related dimer. This complex of two symmetry-related dimers forms a tetramer that reveals novel mechanisms for allosteric regulation and has many features associated with full-length holoenzyme. A model of the tetrameric holoenzyme, based on this structure, is consistent with previous small angle X-ray and neutron scattering data, and is validated with new SAXS data and with an RIα mutation localized to a novel interface unique to the tetramer.
authors.affiliation: Department of Chemistry and Biochemistry, University of California at San Diego, La Jolla, CA 92093, USA.
authors.forename: Angela J
authors.initials: AJ
authors.lastname: Boettcher
authors.forename: Jian
authors.initials: J
authors.lastname: Wu
authors.forename: Choel
authors.initials: C
authors.lastname: Kim
authors.forename: Jie
authors.initials: J
authors.lastname: Yang
authors.forename: Jessica
authors.initials: J
authors.lastname: Bruystens
authors.forename: Nikki
authors.initials: N
authors.lastname: Cheung
authors.forename: Juniper K
authors.initials: JK
authors.lastname: Pennypacker
authors.forename: Donald A
authors.initials: DA
authors.lastname: Blumenthal
authors.forename: Alexandr P
authors.initials: AP
authors.lastname: Kornev
authors.forename: Susan S
authors.initials: SS
authors.lastname: Taylor
mesh_terms: D000494:Allosteric Regulation; D000595:Amino Acid Sequence; D000818:Animals; D020134:Catalytic Domain; D000242:Cyclic AMP / Q000378:metabolism*; D054756:Cyclic AMP-Dependent Protein Kinase RIalpha Subunit / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D004789:Enzyme Activation; D015870:Gene Expression; D020035:Holoenzymes / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D007700:Kinetics; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009154:Mutation; D011485:Protein Binding; D011487:Protein Conformation; D055503:Protein Multimerization; D021122:Protein Subunits / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D053838:Scattering, Small Angle; D017386:Sequence Homology, Amino Acid; D014961:X-Ray Diffraction
chemical_list: D054756:Cyclic AMP-Dependent Protein Kinase RIalpha Subunit; D020035:Holoenzymes; D021122:Protein Subunits; D011994:Recombinant Proteins; D000242:Cyclic AMP