Conformational differences among solution structures of the type Ialpha, IIalpha and IIbeta protein kinase A regulatory subunit homodimers: role of the linker regions

The regulatory (R) subunits of the cAMP-dependent protein kinase (protein kinase A or PKA) are multi-domain proteins responsible for conferring cAMP-dependence and localizing PKA to specific subcellular locations. There are four isoforms of the R subunit in mammals that are similar in molecular mass and domain organization, but clearly serve different biological functions. Although high-resolution structures are available for the cAMP-binding domains and dimerization/docking domains of two isoforms, there are no high-resolution structures of any of the intact R subunit homodimer isoforms. The results of small-angle X-ray scattering studies presented here indicate that the RIalpha, RIIalpha, and RIIbeta homodimers differ markedly in overall shape, despite extensive sequence homology and similar molecular masses. The RIIalpha and RIIbeta homodimers have very extended, rod-like shapes, whereas the RIalpha homodimer likely has a compact Y-shape. Based on a comparison of the R subunit sequences, we predict that the linker regions are the likely cause of these large differences in shape among the isoforms. In addition, we show that cAMP binding does not cause large conformational changes in type Ialpha or IIalpha R subunit homodimers, suggesting that the activation of PKA by cAMP involves only local conformational changes in the R subunits.     

Solution scattering reveals large differences in the global structures of type II protein kinase A isoforms

Isoform diversity within the protein kinase A (PKA) family is achieved by catalytic (C) subunits binding to different isoforms of regulatory subunit homodimers (R2). In a previous small-angle X-ray scattering study, we showed that the type Ialpha R2 homodimer has a distinctive Y-shaped structure, while the IIalpha and IIbeta homodimers are highly flexible and extended in solution. Here we present the results of X-ray scattering experiments on different isoforms of the PKA holoenzyme (R2C2) and show that the type IIbeta R2 homodimer undergoes a dramatic compaction upon binding C subunits that involves a 10A reduction in radius of gyration (from 56 to 46 A) and a 35 A shortening of the maximum linear dimension (from 180-145 A). In contrast, the type IIalpha R2 homodimer shows very little change in these structural parameters and remains extended upon C-subunit binding. This large difference is surprising given the highly conserved sequence and domain organization for the different R isoforms. A mutant RIIbeta holoenzyme and an RIIalpha/RIIbeta chimera were used to explore the role of the sequence linking different functional domains within RIIbeta in the observed C subunit-induced compaction. Structural modeling was used to aid in interpreting the scattering results in terms of the role of inter-domain and inter-subunit contacts in determining the global conformations of the different isoforms. The results provide an important structural foundation for understanding isoform-specific PKA localization and signaling.     

C subunits binding to the protein kinase A RI alpha dimer induce a large conformational change

We present structural data on the RI alpha isoform of the cAMP-dependent protein kinase A that reveal, for the first time, a large scale conformational change within the RI alpha homodimer upon catalytic subunit binding. This result infers that the inhibition of catalytic subunit activity is not the result of a simple docking process but rather is a multi-step process involving local conformational changes both in the cAMP-binding domains as well as in the linker region of the regulatory subunit that impact the global structure of the regulatory homodimer. The results were obtained using small-angle neutron scattering with contrast variation and deuterium labeling. From these experiments we derived information on the shapes and dispositions of the catalytic subunits and regulatory homodimer within a holoenzyme reconstituted with a deuterated regulatory subunit. The scattering data also show that, despite extensive sequence homology between the isoforms, the overall structure of the type I alpha holoenzyme is significantly more compact than the type II alpha isoform. We present a model of the type I alpha holoenzyme, built using available high-resolution structures of the component subunits and domains, which best fits the neutron-scattering data. In this model, the type I alpha holoenzyme forms a flattened V shape with the RI alpha dimerization domain at the point of the V and the cAMP-binding domains of the RI alpha subunits with their bound catalytic subunits at the ends.     

R-subunit isoform specificity in protein kinase A: distinct features of protein interfaces in PKA types I and II by amide H/2H exchange mass spectrometry

The two isoforms (RI and RII) of the regulatory (R) subunit of cAMP-dependent protein kinase or protein kinase A (PKA) are similar in sequence yet have different biochemical properties and physiological functions. To further understand the molecular basis for R-isoform-specificity, the interactions of the RIIβ isoform with the PKA catalytic (C) subunit were analyzed by amide H/²H exchange mass spectrometry to compare solvent accessibility of RIIβ and the C subunit in their free and complexed states. Direct mapping of the RIIβ-C interface revealed important differences between the intersubunit interfaces in the type I and type II holoenzyme complexes. These differences are seen in both the R-subunits as well as the C-subunit. Unlike the type I isoform, the type II isoform complexes require both cAMP-binding domains, and ATP is not obligatory for high affinity interactions with the C-subunit. Surprisingly, the C-subunit mediates distinct, overlapping surfaces of interaction with the two R-isoforms despite a strong homology in sequence and similarity in domain organization. Identification of a remote allosteric site on the C-subunit that is essential for interactions with RII, but not RI subunits, further highlights the considerable diversity in interfaces found in higher order protein complexes mediated by the C-subunit of PKA.     

Consequences of cAMP and catalytic-subunit binding on the flexibility of the A-kinase regulatory subunit

A combination of site-directed labeling and time-resolved fluorescence anisotropy was used to further elucidate the structure and underlying dynamic features of the type I regulatory (R(I)(alpha)) subunit of the cAMP-dependent protein kinase. Specifically, the consequences of cAMP and the catalytic (C)-subunit binding on the backbone flexibility around seven sites of cysteine substitution and fluorescein maleimide labeling (Thr(6)Cys, Leu(66)Cys, Ser(75)Cys, Ser(81)Cys, Ser(99)Cys, Ser(145)Cys, and Ser(373)Cys) in the R(I)(alpha) subunit were assessed. Focusing on the fast rotational correlation time, the results indicate that most of the interdomain segment connecting the dimerization/docking (D/D) and tandem cAMP-binding domains is probably weakly associated with the latter domain. Also, this segment becomes more tightly bound to the C subunit upon holoenzyme formation. The results also suggest that there is a short 'hinge' segment (around Leu(66)Cys) that could allow the structured interdomain/cAMP-binding and D/D domains to pivot about each other. Finally, cAMP binding dramatically reduces the backbone flexibility around only the two sites of cysteine substitution in the cAMP-binding domains, suggesting a selective structural stabilization caused by cAMP and a "tight" coupling of low-nanosecond fluctuations selectively within the tandem cAMP-binding domains.     

Rearrangements in a hydrophobic core region mediate cAMP action in the regulatory subunit of PKA

cAMP-dependent protein kinase (PKA) forms an inactive heterotetramer of two regulatory (R; with two cAMP-binding domains A and B each) and two catalytic (C) subunits. Upon the binding of four cAMP molecules to the R dimer, the monomeric C subunits dissociate. Based on sequence analysis of cyclic nucleotide-binding domains in prokaryotes and eukaryotes and on crystal structures of cAMP-bound R subunit and cyclic nucleotide-free Epac (exchange protein directly activated by cAMP), four amino acids were identified (Leu203, Tyr229, Arg239 and Arg241) and probed for cAMP binding to the R subunits and for R/C interaction. Arg239 and Arg241 (mutated to Ala and Glu) displayed no differences in the parameters investigated. In contrast, Leu203 (mutated to Ala and Trp) and Tyr229 (mutated to Ala and Thr) exhibited up to 30-fold reduced binding affinity for the C subunit and up to 120-fold reduced binding affinity for cAMP. Tyr229Asp showed the most severe effects, with 350-fold reduced affinity for cAMP and no detectable binding to the C subunit. Based on these results and structural data in the cAMP-binding domain, a switch mechanism via a hydrophobic core region is postulated that is comparable to an activation model proposed for Epac.     

Mapping intersubunit interactions of the regulatory subunit (RIalpha) in the type I holoenzyme of protein kinase A by amide hydrogen/deuterium exchange mass spectrometry (DXMS)

Protein kinase A (PKA), a central locus for cAMP signaling in the cell, is composed of regulatory (R) and catalytic (C) subunits. The C-subunits are maintained in an inactive state by binding to the R-subunit dimer in a tetrameric holoenzyme complex (R(2)C(2)). PKA is activated by cAMP binding to the R-subunits which induces a conformational change leading to release of the active C-subunit. Enzymatic activity of the C-subunit is thus regulated by cAMP via the R-subunit, which toggles between cAMP and C-subunit bound states. The R-subunit is composed of a dimerization/docking (D/D) domain connected to two cAMP-binding domains (cAMP:A and cAMP:B). While crystal structures of the free C-subunit and cAMP-bound states of a deletion mutant of the R-subunit are known, there is no structure of the holoenzyme complex or of the cAMP-free state of the R-subunit. An important step in understanding the cAMP-dependent activation of PKA is to map the R-C interface and characterize the mutually exclusive interactions of the R-subunit with cAMP and C-subunit. Amide hydrogen/deuterium exchange mass spectrometry is a suitable method that has provided insights into the different states of the R-subunit in solution, thereby allowing mapping of the effects of cAMP and C-subunit on different regions of the R-subunit. Our study has localized interactions with the C-subunit to a small contiguous surface on the cAMP:A domain and the linker region. In addition, C-subunit binding causes increased amide hydrogen exchange within both cAMP-domains, suggesting that these regions become more flexible in the holoenzyme and are primed to bind cAMP. Furthermore, the difference in the protection patterns between RIalpha and the previously studied RIIbeta upon cAMP-binding suggests isoform-specific differences in cAMP-dependent regulation of PKA activity.     

PKA-I holoenzyme structure reveals a mechanism for cAMP-dependent activation

Protein kinase A (PKA) holoenzyme is one of the major receptors for cyclic adenosine monophosphate (cAMP), where an extracellular stimulus is translated into a signaling response. We report here the structure of a complex between the PKA catalytic subunit and a mutant RI regulatory subunit, RIalpha(91-379:R333K), containing both cAMP-binding domains. Upon binding to the catalytic subunit, RI undergoes a dramatic conformational change in which the two cAMP-binding domains uncouple and wrap around the large lobe of the catalytic subunit. This large conformational reorganization reveals the concerted mechanism required to bind and inhibit the catalytic subunit. The structure also reveals a holoenzyme-specific salt bridge between two conserved residues, Glu261 and Arg366, that tethers the two adenine capping residues far from their cAMP-binding sites. Mutagenesis of these residues demonstrates their importance for PKA activation. Our structural insights, combined with the mutagenesis results, provide a molecular mechanism for the ordered and cooperative activation of PKA by cAMP.     

A point mutation abolishes binding of cAMP to site A in the regulatory subunit of cAMP-dependent protein kinase

Each regulatory subunit of cAMP-dependent protein kinase has two tandem cAMP-binding sites, A and B, at the carboxyl terminus. Based on sequence homologies with the cAMP-binding domain of the Escherichia coli catabolite gene activator protein, a model has been constructed for each cAMP-binding domain. Two of the conserved features of each cAMP-binding site are an arginine and a glutamic acid which interact with the negatively charged phosphate and with the 2'-OH on the ribose ring, respectively. In the type I regulatory subunit, this arginine in cAMP binding site A is Arg-209. Recombinant DNA techniques have been used to change this arginine to a lysine. The resulting protein binds cAMP with a high affinity and associates with the catalytic subunit to form holoenzyme. The mutant holoenzyme also is activated by cAMP. However, the mutant R-subunit binds only 1 mol of cAMP/R-monomer. Photoaffinity labeling confirmed that the mutant R-subunit has only one functional cAMP-binding site. In contrast to the native R-subunit which is labeled at Trp-260 and Tyr-371 by 8-N3cAMP, the mutant R-subunit is convalently modified at a single site, Tyr-371, which correlates with a functional cAMP-binding site B. The lack of functional cAMP-binding site A also was confirmed by activating the mutant holoenzyme with analogs of cAMP which have a high specificity for either site A or site B. 8-NH2-methyl cAMP which preferentially binds to site B was similar to cAMP in its ability to activate both mutant and wild type holoenzyme whereas N6-monobutyryl cAMP, a site A-specific analog, was a very poor activator of the mutant holoenzyme. The results support the conclusions that 1) Arg-209 is essential for cAMP binding to site A and 2) cAMP binding to domain A is not essential for dissociation of the mutant holoenzyme.     

Phosphodiesterases catalyze hydrolysis of cAMP-bound to regulatory subunit of protein kinase A and mediate signal termination

Although extensive structural and biochemical studies have provided molecular insights into the mechanism of cAMP-dependent activation of protein kinase A (PKA), little is known about signal termination and the role of phosphodiesterases (PDEs) in regulatory feedback. In this study we describe a novel mode of protein kinase A-anchoring protein (AKAP)-independent feedback regulation between a specific PDE, RegA and the PKA regulatory (RIα) subunit, where RIα functions as an activator of PDE catalysis. Our results indicate that RegA, in addition to its well-known role as a PDE for bulk cAMP in solution, is also capable of hydrolyzing cAMP-bound to RIα. Furthermore our results indicate that binding of RIα activates PDE catalysis several fold demonstrating a dual function of RIα, both as an inhibitor of the PKA catalytic (C) subunit and as an activator for PDEs. Deletion mutagenesis has localized the sites of interaction to one of the cAMP-binding domains of RIα and the catalytic PDE domain of RegA whereas amide hydrogen/deuterium exchange mass spectrometry has revealed that the cAMP-binding site (phosphate binding cassette) along with proximal regions important for relaying allosteric changes mediated by cAMP, are important for interactions with the PDE catalytic domain of RegA. These sites of interactions together with measurements of cAMP dissociation rates demonstrate that binding of RegA facilitates dissociation of cAMP followed by hydrolysis of the released cAMP to 5'AMP. cAMP-free RIα generated as an end product remains bound to RegA. The PKA C-subunit then displaces RegA and reassociates with cAMP-free RIα to regenerate the inactive PKA holoenzyme thereby completing the termination step of cAMP signaling. These results reveal a novel mode of regulatory feedback between PDEs and RIα that has important consequences for PKA regulation and cAMP signal termination.     

cAMP-dependent protein kinase regulatory subunit type IIbeta: active site mutations define an isoform-specific network for allosteric signaling by cAMP

cAMP-dependent protein kinase (cAPK) contains a regulatory (R) subunit dimer bound to two catalytic (C) subunits. Each R monomer contains two cAMP-binding domains, designated A and B. The sequential binding of two cAMPs releases active C. We describe here the properties of RIIbeta and two mutant RIIbeta subunits, engineered by converting a conserved Arg to Lys in each cAMP-binding domain thereby yielding a protein that contains one intact, high affinity cAMP-binding site and one defective site. Structure and function were characterized by circular dichroism, steady-state fluorescence, surface plasmon resonance and holoenzyme activation assays. The Ka for RIIbeta is 610 nM, which is 10-fold greater than its Kd(cAMP) and significantly higher than for RIalpha and RIIalpha. The Arg mutant proteins demonstrate that the conserved Arg is important for both cAMP binding and organization of each domain and that binding to domain A is required for activation. The Ka of the A domain mutant protein is 21-fold greater than that of wild-type and the Kd(cAMP) is increased 7-fold, confirming that cAMP must bind to the mutated site to initiate activation. The domain B mutant Ka is 2-fold less than its Kd(cAMP), demonstrating that, unlike RIalpha, cAMP can access the A site even when the B site is empty. Removal of the B domain yields a Ka identical to the Kd(cAMP) of full-length RIIbeta, indicating that the B domain inhibits holoenzyme activation for RIIbeta. In RIalpha, removal of the B domain generates a protein that is more difficult to activate than the wild-type protein.     

Differential binding of cAMP-dependent protein kinase regulatory subunit isoforms Ialpha and IIbeta to the catalytic subunit

Limited trypsin digestion of type I cAMP-dependent protein kinase holoenzyme results in a proteolytic-resistant Delta(1-72) regulatory subunit core, indicating that interaction between the regulatory and catalytic subunits extends beyond the autoinhibitory site in the R subunit at the NH(2) terminus. Sequence alignment of the two R subunit isoforms, RI and RII, reveals a significantly sequence diversity at this specific region. To determine whether this sequence diversity is functionally important for interaction with the catalytic subunit, specific mutations, R133A and D328A, are introduced into sites adjacent to the active site cleft in the catalytic subunit. While replacing Arg(133) with Ala decreases binding affinity for RII, interaction between the catalytic subunit and RI is not affected. In contrast, mutant C(D328A) showed a decrease in affinity for binding RI while maintaining similar affinities for RII as compared with the wild-type catalytic subunit. These results suggest that sequence immediately NH(2)-terminal to the consensus inhibition site in RI and RII interacts with different sites at the proximal region of the active site cleft in the catalytic subunit. These isoform-specific differences would dictate a significantly different domain organization in the type I and type II holoenzymes.     

Interactions between regulatory and catalytic subunits of the Candida albicans cAMP-dependent protein kinase are modulated by autophosphorylation of the regulatory subunit

The cAMP-dependent protein kinase (PKA) from Candida albicans is a tetramer composed of two catalytic subunits (C) and two type II regulatory subunits (R). To evaluate the role of a putative autophosphorylation site of the R subunit (Ser(180)) in the interaction with C, this site was mutated to an Ala residue. Recombinant wild-type and mutant forms of the R subunit were expressed in Escherichia coli and purified. The wild-type recombinant R subunit was fully phosphorylated by the purified C subunit, while the mutant form was not, confirming that Ser(180) is the target for the autophosphorylation reaction. Association and dissociation experiments conducted with both recombinant R subunits and purified C subunit showed that intramolecular phosphorylation of the R subunit led to a decreased affinity for C. This diminished affinity was reflected by an 8-fold increase in the concentration of R subunit needed to reach half-maximal inhibition of the kinase activity and in a 5-fold decrease in the cAMP concentration necessary to obtain half-maximal dissociation of the reconstituted holoenzyme. Dissociation of the mutant holoenzyme by cAMP was not affected by the presence of MgATP. Metabolic labeling of yeast cells with [(32)P]orthophosphate indicated that the R subunit exists as a serine phosphorylated protein. The possible involvement of R subunit autophosphorylation in modulating C. albicans PKA activity in vivo is discussed.     

Probing cAMP-dependent protein kinase holoenzyme complexes I alpha and II beta by FT-IR and chemical protein footprinting

Although individual structures of cAMP-dependent protein kinase (PKA) catalytic (C) and regulatory (R) subunits have been determined at the atomic level, our understanding of the effects of cAMP activation on protein dynamics and intersubunit communication of PKA holoenzymes is very limited. To delineate the mechanism of PKA activation and structural differences between type I and II PKA holoenzymes, the conformation and structural dynamics of PKA holoenzymes Ialpha and IIbeta were probed by amide hydrogen-deuterium exchange coupled with Fourier transform infrared spectroscopy (FT-IR) and chemical protein footprinting. Binding of cAMP to PKA holoenzymes Ialpha and IIbeta leads to a downshift in the wavenumber for both the alpha-helix and beta-strand bands, suggesting that R and C subunits become overall more dynamic in the holoenzyme complexes. This is consistent with the H-D exchange results showing a small change in the overall rate of exchange in response to the binding of cAMP to both PKA holoenzymes Ialpha and IIbeta. Despite the overall similarity, significant differences in the change of FT-IR spectra in response to the binding of cAMP were observed between PKA holoenzymes Ialpha and IIbeta. Activation of PKA holoenzyme Ialpha led to more conformational changes in beta-strand structures, while cAMP induced more apparent changes in the alpha-helical structures in PKA holoenzyme IIbeta. Chemical protein footprinting experiments revealed an extended docking surface for the R subunits on the C subunit. Although the overall subunit interfaces appeared to be similar for PKA holoenzymes Ialpha and IIbeta, a region around the active site cleft of the C subunit was more protected in PKA holoenzyme Ialpha than in PKA holoenzyme IIbeta. These results suggest that the C subunit assumes a more open conformation in PKA holoenzyme IIbeta. In addition, the chemical cleavage patterns around the active site cleft of the C subunit were distinctly different in PKA holoenzymes Ialpha and IIbeta even in the presence of cAMP. These observations provide direct evidence that the R subunits may be partially associated with the C subunit with the pseudosubstrate sequence docked in the active site cleft in the presence of cAMP.     

Sensing Domain Dynamics in Protein Kinase A-I�� Complexes by Solution X-ray Scattering

The catalytic (C) and regulatory (R) subunits of protein kinase A are exceptionally dynamic proteins. Interactions between the R- and C-subunits are regulated by cAMP binding to the two cyclic nucleotide-binding domains in the R-subunit. Mammalian cells express four different isoforms of the R-subunit (RIα, RIβ, RIIα, and RIIβ) that all interact with the C-subunit in different ways. Here, we investigate the dynamic behavior of protein complexes between RIα and C-subunits using small angle x-ray scattering. We show that a single point mutation in RIα, R333K (which alters the cAMP-binding properties of Domain B) results in a compact shape compared with the extended shape of the wild-type R·C complex. A double mutant complex that disrupts the interaction site between the C-subunit and Domain B in RIα, RIα_ABR333K·C(K285P), results in a broader P(r) curve that more closely resembles the P(r) profiles of wild-type complexes. These results together suggest that interactions between RIα Domain B and the C-subunit in the RIα·C complex involve large scale dynamics that can be disrupted by single point mutations in both proteins. In contrast to RIα·C complexes. Domain B in the RIIβ·C heterodimer is not dynamic and is critical for both inhibition and complex formation. Our study highlights the functional differences of domain dynamics between protein kinase A isoforms, providing a framework for elucidating the global organization of each holoenzyme and the cross-talk between the R- and C-subunits.     

Coordinate pretranslational control of cAMP-dependent protein kinase subunit expression during development in the water mold Blastocladiella emersonii.

The aquatic fungus Blastocladiella emersonii provides a system for studying the regulation of expression of regulatory (R) and catalytic (C) subunits of cAMP-dependent protein kinase (PKA). Blastocladiella cells contain a single PKA with properties very similar to type II kinases of mammalian tissues. During development cAMP-dependent protein kinase activity and its associated cAMP-binding activity change drastically. We have previously shown that the increase in cAMP-binding activity during sporulation is due to de novo synthesis of R subunit and to an increase in the translatable mRNA coding for R (Marques et al., Eur. J. Biochem. 178, 803, 1989). In the present work we have continued these studies to investigate the mechanism by which the changes in the level of kinase activity take place. The C subunit of Blastocladiella has been purified; antiserum has been raised against it and used to determine amounts of C subunit throughout the fungus' life cycle. A sharp increase in C subunit content occurs during sporulation and peaks at the zoospore stage. Northern blot analyses, using Blastocladiella C and R cDNA probes, have shown that the levels of C and R mRNAs parallel their intracellular protein concentrations. These results indicate a coordinate pretranslational control for C and R subunit expression during differentiation in Blastocladiella.     

Realizing the allosteric potential of the tetrameric protein kinase A RIα holoenzyme

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.     

Amide H/2H exchange reveals communication between the cAMP and catalytic subunit-binding sites in the R(I)alpha subunit of protein kinase A

The changes in backbone hydrogen/deuterium (H/2H) exchange in the regulatory subunit (R(I)alpha(94-244)) of cyclic AMP-dependent protein kinase A (PKA) were probed by MALDI-TOF mass spectrometry. The three naturally occurring states of the regulatory subunit were studied: (1) free R(I)alpha(94-244), which likely represents newly synthesized protein, (2) R(I)alpha(94-244) bound to the catalytic (C) subunit, or holoenzyme, and (3) R(I)alpha(94-244) bound to cAMP. Protection from amide exchange upon C-subunit binding was observed for the helical subdomain, including the A-helix and B-helix, pointing to regions adjacent to those shown to be important by mutagenesis. In addition, C-subunit binding caused changes in observed amide exchange in the distal cAMP-binding pocket. Conversely, cAMP binding caused protection in the cAMP-binding pocket and increased exchange in the helical subdomain. These results suggest that the mutually exclusive binding of either cAMP or C-subunit is controlled by binding at one site transmitting long distance changes to the other site.     

Expression and characterization of mutant forms of the type I regulatory subunit of cAMP-dependent protein kinase. The effect of defective cAMP binding on holoenzyme activation

The mouse wild type and four mutant regulatory type I (RI) subunits were expressed in Escherichia coli and subjected to kinetic analyses. The defective RI subunits had point mutations in either cAMP-binding site A (G200/E), site B (G324/D, R332/H), or in both binding sites. In addition, a truncated form of RI which lacked the entire cAMP-binding site B was generated. All of the mutant RI subunits which bound [3H]cAMP demonstrated more rapid rates of cAMP dissociation compared to the wild type RI subunit. Dissociation profiles showed only a single dissociation component, suggesting that a single nonmutated binding site was functional. The mutant RI subunits associated with purified native catalytic subunit to form chromatographically separable holoenzyme complexes in which catalytic activity was suppressed. Each of these holoenzymes could be activated but showed varying degrees of cAMP responsiveness with apparent Ka values ranging from 40 nM to greater than 5 microM. The extent to which the mutated cAMP-binding sites were defective was also shown by the resistance of the respective holoenzymes to activation by cAMP analogs selective for the mutated binding sites. Kinetic results support the conclusions that 1) Gly-200 of cAMP-binding site A and Gly-324 or Arg-332 of site B are essential to normal conformation and function, 2) activation of type I cAMP-dependent protein kinase requires that only one of the cAMP-binding sites be functional, 3) mutational inactivation of site B (slow exchange) has a much more drastic effect than that of site A on increasing the Ka of the holoenzyme for cAMP, as well as in altering the rate of cAMP dissociation from the remaining site of the free RI subunit. The strong dependence of one cAMP-binding site on the integrity of the other site suggests a tight association between the two sites.     

Dynamic binding of PKA regulatory subunit RI alpha

Recent crystal structures have revealed that regulatory subunit RIalpha of PKA undergoes a dramatic conformational change upon complex formation with the catalytic subunit. Molecular dynamics studies were initiated to elucidate the contributions of intrinsic conformational flexibility and interactions with the catalytic subunit in formation and stabilization of the complex. Simulations of a single RIalpha nucleotide binding domain (NBD), missing cAMP, showed that its C helix spontaneously occupies two distinct conformations: either packed against the nucleotide binding domain as in its cAMP bound structure, or extended into an intermediate form resembling that of the holoenzyme structure. C helix extension was not seen in a simulation of either RIalpha NBD. In a model complex containing both NBDs and the catalytic subunit, well-conserved residues at the interface between the NBDs in the cAMP bound form were found to stabilize the complex through contacts with the catalytic subunit. The model structure is consistent with available experimental data.