ABSTRACT
Protein kinase A (PKA) is ubiquitously expressed and is responsible for regulating many important cellular functions in response to changes in intracellular cAMP concentrations. The PKA holoenzyme is a tetramer (R2:C2), with a regulatory subunit homodimer (R2) that binds and inhibits two catalytic (C) subunits; binding of cAMP to the regulatory subunit homodimer causes activation of the catalytic subunits. Four different R subunit isoforms exist in mammalian cells, and these confer different structural features, subcellular localization, and biochemical properties upon the PKA holoenzymes they form. The holoenzyme containing RIIß is structurally unique in that the type IIß holoenzyme is much more compact than the free RIIß homodimer. We have used small angle x-ray scattering and small angle neutron scattering to study the solution structure and subunit organization of a holoenzyme containing an RIIß C-terminal deletion mutant (RIIß(1-280)), which is missing the C-terminal cAMP-binding domain to better understand the structural organization of the type IIß holoenzyme and the RIIß domains that contribute to stabilizing the holoenzyme conformation. Our results demonstrate that compaction of the type IIß holoenzyme does not require the C-terminal cAMP-binding domain but rather involves large structural rearrangements within the linker and N-terminal cyclic nucleotide-binding domain of the RIIß homodimer. The structural rearrangements are significantly greater than seen previously with RIIα and are likely to be important in mediating short range and long range interdomain and intersubunit interactions that uniquely regulate the activity of the type IIß isoform of PKA.
Subject(s)
Cyclic AMP-Dependent Protein Kinase Catalytic Subunits/chemistry , Cyclic AMP-Dependent Protein Kinase RIIbeta Subunit/chemistry , Cyclic AMP/chemistry , Holoenzymes/chemistry , Animals , Catalytic Domain , Cyclic AMP/metabolism , Cyclic AMP-Dependent Protein Kinase Catalytic Subunits/genetics , Cyclic AMP-Dependent Protein Kinase Catalytic Subunits/metabolism , Cyclic AMP-Dependent Protein Kinase RIIbeta Subunit/genetics , Cyclic AMP-Dependent Protein Kinase RIIbeta Subunit/metabolism , Escherichia coli/genetics , Escherichia coli/metabolism , Gene Expression , Holoenzymes/genetics , Holoenzymes/metabolism , Isoenzymes/chemistry , Isoenzymes/genetics , Isoenzymes/metabolism , Mice , Models, Molecular , Mutation , Neutron Diffraction , Protein Binding , Protein Structure, Secondary , Protein Structure, Tertiary , Rats , Recombinant Proteins/chemistry , Recombinant Proteins/genetics , Recombinant Proteins/metabolism , Scattering, Small Angle , X-Ray DiffractionABSTRACT
In its physiological state, cyclic adenosine monophosphate (cAMP)-dependent protein kinase (PKA) is a tetramer that contains a regulatory (R) subunit dimer and two catalytic (C) subunits. We describe here the 2.3 angstrom structure of full-length tetrameric RIIß(2):C(2) holoenzyme. This structure showing a dimer of dimers provides a mechanistic understanding of allosteric activation by cAMP. The heterodimers are anchored together by an interface created by the ß4-ß5 loop in the RIIß subunit, which docks onto the carboxyl-terminal tail of the adjacent C subunit, thereby forcing the C subunit into a fully closed conformation in the absence of nucleotide. Diffusion of magnesium adenosine triphosphate (ATP) into these crystals trapped not ATP, but the reaction products, adenosine diphosphate and the phosphorylated RIIß subunit. This complex has implications for the dissociation-reassociation cycling of PKA. The quaternary structure of the RIIß tetramer differs appreciably from our model of the RIα tetramer, confirming the small-angle x-ray scattering prediction that the structures of each PKA tetramer are different.
Subject(s)
Cyclic AMP-Dependent Protein Kinase Catalytic Subunits/chemistry , Cyclic AMP-Dependent Protein Kinase Catalytic Subunits/metabolism , Cyclic AMP-Dependent Protein Kinase RIIbeta Subunit/chemistry , Cyclic AMP-Dependent Protein Kinase RIIbeta Subunit/metabolism , Adenosine Triphosphate/metabolism , Allosteric Regulation , Allosteric Site , Amino Acid Sequence , Animals , Binding Sites , Crystallization , Crystallography, X-Ray , Cyclic AMP/metabolism , Holoenzymes/chemistry , Holoenzymes/metabolism , Hydrophobic and Hydrophilic Interactions , Mice , Models, Molecular , Molecular Sequence Data , Mutant Proteins/chemistry , Mutant Proteins/metabolism , Protein Binding , Protein Folding , Protein Multimerization , Protein Structure, Quaternary , Protein Structure, Tertiary , RatsABSTRACT
Cyclic GMP-dependent protein kinase (PKG) is a key mediator of the nitric oxide/cGMP signaling pathway and plays a central role in regulating cardiovascular and neuronal functions. The N-terminal â¼50 amino acids of the kinase are required for homodimerization and association with isoform-specific PKG-anchoring proteins (GKAPs), which target the kinase to specific substrates. To understand the molecular details of PKG dimerization and gain insight into its association with GKAPs, we solved a crystal structure of the PKG Iß dimerization/docking domain. Our structure provides molecular details of this unique leucine/isoleucine zipper, revealing specific hydrophobic and ionic interactions that mediate dimerization and demonstrating the topology of the GKAP interaction surface.