Dynamics of cAPK type IIbeta activation revealed by enhanced amide H/2H exchange mass spectrometry (DXMS)

cAMP-dependent protein kinase (cAPK) is a key component in numerous cell signaling pathways. The cAPK regulatory (R) subunit maintains the kinase in an inactive state until cAMP saturation of the R-subunit leads to activation of the enzyme. To delineate the conformational changes associated with cAPK activation, the amide hydrogen/deuterium exchange in the cAPK type IIbeta R-subunit was probed by electrospray mass spectrometry. Three states of the R-subunit, cAMP-bound, catalytic (C)-subunit bound, and apo, were incubated in deuterated water for various lengths of time and then, prior to mass spectrometry analysis, subjected to digestion by pepsin to localize the deuterium incorporation. High sequence coverage (>99%) by the pepsin-digested fragments enables us to monitor the dynamics of the whole protein. The effects of cAMP binding on RIIbeta amide hydrogen exchange are restricted to the cAMP-binding pockets, while the effects of C-subunit binding are evident across both cAMP-binding domains and the linker region. The decreased amide hydrogen exchange for residues 253-268 within cAMP binding domain A and for residues 102-115, which include the pseudosubstrate inhibitory site, support the prediction that these two regions represent the conserved primary and peripheral C-subunit binding sites. An increase in amide hydrogen exchange for a broad area within cAMP-binding domain B and a narrow area within cAMP-binding domain A (residues 222-232) suggest that C-subunit binding transmits long-distance conformational changes throughout the protein.     

Mechanism of intracellular cAMP sensor Epac2 activation: cAMP-induced conformational changes identified by amide hydrogen/deuterium exchange mass spectrometry (DXMS)

Epac2, a guanine nucleotide exchange factor, regulates a wide variety of intracellular processes in response to second messenger cAMP. In this study, we have used peptide amide hydrogen/deuterium exchange mass spectrometry to probe the solution structural and conformational dynamics of full-length Epac2 in the presence and absence of cAMP. The results support a mechanism in which cAMP-induced Epac2 activation is mediated by a major hinge motion centered on the C terminus of the second cAMP binding domain. This conformational change realigns the regulatory components of Epac2 away from the catalytic core, making the later available for effector binding. Furthermore, the interface between the first and second cAMP binding domains is highly dynamic, providing an explanation of how cAMP gains access to the ligand binding sites that, in the crystal structure, are seen to be mutually occluded by the other cAMP binding domain. Moreover, cAMP also induces conformational changes at the ionic latch/hairpin structure, which is directly involved in RAP1 binding. These results suggest that in addition to relieving the steric hindrance imposed upon the catalytic lobe by the regulatory lobe, cAMP may also be an allosteric modulator directly affecting the interaction between Epac2 and RAP1. Finally, cAMP binding also induces significant conformational changes in the dishevelled/Egl/pleckstrin (DEP) domain, a conserved structural motif that, although missing from the active Epac2 crystal structure, is important for Epac subcellular targeting and in vivo functions.     

Search for new cyclic AMP-binding proteins

Today, there is evidence that the cAMP-dependent kinases (PKA) are not the only intracellular receptors involved in intracellular cAMP signalling in eukaryotes. Other cAMP-binding proteins have been recently identified, including some cyclic nucleotide-gated channels and Epac (exchange protein directly activated by cAMP) proteins. All these proteins bind cAMP through conserved cyclic nucleotide monophosphate-binding domains. However, all putative cAMP-binding proteins having such domains, as revealed by computer analysis, do not necessarily bind cAMP, indicating that their presence is not a sufficient criteria to predict cAMP-binding property for a protein.     

Dissecting interdomain communication within cAPK regulatory subunit type IIbeta using enhanced amide hydrogen/deuterium exchange mass spectrometry (DXMS)

cAMP-dependent protein kinase (cAPK) is a heterotetramer containing a regulatory (R) subunit dimer bound to two catalytic (C) subunits and is involved in numerous cell signaling pathways. The C-subunit is activated allosterically when two cAMP molecules bind sequentially to the cAMP-binding domains, designated A and B (cAB-A and cAB-B, respectively). Each cAMP-binding domain contains a conserved Arg residue that is critical for high-affinity cAMP binding. Replacement of this Arg with Lys affects cAMP affinity, the structural integrity of the cAMP-binding domains, and cAPK activation. To better understand the local and long-range effects that the Arg-to-Lys mutation has on the dynamic properties of the R-subunit, the amide hydrogen/deuterium exchange in the RIIbeta subunit was probed by electrospray mass spectrometry. Mutant proteins containing the Arg-to-Lys substitution in either cAMP-binding domain were deuterated for various times and then, prior to mass spectrometry analysis, subjected to pepsin digestion to localize the deuterium incorporation. Mutation of this Arg in cAB-A (Arg230) causes an increase in amide hydrogen exchange throughout the mutated domain that is beyond the modest and localized effects of cAMP removal and is indicative of the importance of this Arg in domain organization. Mutation of Arg359 (cAB-B) leads to increased exchange in the adjacent cAB-A domain, particularly in the cAB-A domain C-helix that lies on top of the cAB-B domain and is believed to be functionally linked to the cAB-B domain. This interdomain communication appears to be a unidirectional pathway, as mutation of Arg230 in cAB-A does not effect dynamics of the cAB-B domain.     

Cyclic nucleotide selectivity of protein kinase G isozymes

The intrinsic activity of the C‐terminal catalytic (C) domain of cyclic guanosine monophosphate (cGMP)‐dependent protein kinases (PKG) is inhibited by interactions with the N‐terminal regulatory (R) domain. Selective binding of cGMP to cyclic nucleotide binding (CNB) domains within the R‐domain disrupts the inhibitory R–C interaction, leading to the release and activation of the C‐domain. Affinity measurements of mammalian and plasmodium PKG CNB domains reveal different degrees of cyclic nucleotide affinity and selectivity; the CNB domains adjacent to the C‐domain are more cGMP selective and therefore critical for cGMP‐dependent activation. Crystal structures of isolated CNB domains in the presence and absence of cyclic nucleotides reveal isozyme‐specific contacts that explain cyclic nucleotide selectivity and conformational changes that accompany CNB. Crystal structures of tandem CNB domains identify two types of CNB‐mediated dimeric contacts that indicate cGMP‐driven reorganization of domain–domain interfaces that include large conformational changes. Here, we review the available structural and functional information of PKG CNB domains that further advance our understanding of cGMP mediated regulation and activation of PKG isozymes.     

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.     

Allosteric Inhibition of Epac: COMPUTATIONAL MODELING AND EXPERIMENTAL VALIDATION TO IDENTIFY ALLOSTERIC SITES AND INHIBITORS*

Epac, a guanine nucleotide exchange factor for the low molecular weight G protein Rap, is an effector of cAMP signaling and has been implicated to have roles in numerous diseases, including diabetes mellitus, heart failure, and cancer. We used a computational molecular modeling approach to predict potential binding sites for allosteric modulators of Epac and to identify molecules that might bind to these regions. This approach revealed that the conserved hinge region of the cyclic nucleotide-binding domain of Epac1 is a potentially druggable region of the protein. Using a bioluminescence resonance energy transfer-based assay (CAMYEL, cAMP sensor using YFP-Epac-Rluc), we assessed the predicted compounds for their ability to bind Epac and modulate its activity. We identified a thiobarbituric acid derivative, 5376753, that allosterically inhibits Epac activity and used Swiss 3T3 and HEK293 cells to test the ability of this compound to modulate the activity of Epac and PKA, as determined by Rap1 activity and vasodilator-stimulated phosphoprotein phosphorylation, respectively. Compound 5376753 selectively inhibited Epac in biochemical and cell migration studies. These results document the utility of a computational approach to identify a domain for allosteric regulation of Epac and a novel compound that prevents the activation of Epac1 by cAMP.     

cAMP-regulated Protein Lysine Acetylases in Mycobacteria

Cyclic AMP synthesized by Mycobacterium tuberculosis has been shown to play a role in pathogenesis. However, the high levels of intracellular cAMP found in both pathogenic and non-pathogenic mycobacteria suggest that additional and important biological processes are regulated by cAMP in these organisms. We describe here the biochemical characterization of novel cAMP-binding proteins in M. smegmatis and M. tuberculosis (MSMEG_5458 and Rv0998, respectively) that contain a cyclic nucleotide binding domain fused to a domain that shows similarity to the GNAT family of acetyltransferases. We detect protein lysine acetylation in mycobacteria and identify a universal stress protein (USP) as a substrate of MSMEG_5458. Acetylation of a lysine residue in USP is regulated by cAMP, and using a strain deleted for MSMEG_5458, we show that USP is indeed an in vivo substrate for MSMEG_5458. The Rv0998 protein shows a strict cAMP-dependent acetylation of USP, despite a lower affinity for cAMP than MSMEG_5458. Thus, this report not only represents the first demonstration of protein lysine acetylation in mycobacteria but also describes a unique functional interplay between a cyclic nucleotide binding domain and a protein acetyltransferase.     

Binding of cyclic diguanylate in the non-catalytic EAL domain of FimX induces a long-range conformational change

FimX is a multidomain signaling protein required for type IV pilus biogenesis and twitching motility in the opportunistic pathogen Pseudomonas aeruginosa. FimX is localized to the single pole of the bacterial cell, and the unipolar localization is crucial for the correct assembly of type IV pili. FimX contains a non-catalytic EAL domain that lacks cyclic diguanylate (c-di-GMP) phosphodiesterase activity. It was shown that deletion of the EAL domain or mutation of the signature EVL motif affects the unipolar localization of FimX. However, it was not understood how the C-terminal EAL domain could influence protein localization considering that the localization sequence resides in the remote N-terminal region of the protein. Using hydrogen/deuterium exchange-coupled mass spectrometry, we found that the binding of c-di-GMP to the EAL domain triggers a long-range (∼ca. 70 Å) conformational change in the N-terminal REC domain and the adjacent linker. In conjunction with the observation that mutation of the EVL motif of the EAL domain abolishes the binding of c-di-GMP, the hydrogen/deuterium exchange results provide a molecular explanation for the mediation of protein localization and type IV pilus biogenesis by c-di-GMP through a remarkable allosteric regulation mechanism.     

Amide hydrogen exchange reveals conformational changes in hsp70 chaperones important for allosteric regulation

Hsp70 chaperones assist protein folding processes by a nucleotide-driven cycle of substrate binding and release. Although structural information is available for the isolated nucleotide-binding (NBD) and substrate-binding domains (SBD) in the high affinity conformation, the low affinity conformations and the conformational changes associated with mutual allosteric regulation remained largely enigmatic. By using amide hydrogen exchange in combination with mass spectrometry, we analyzed the Escherichia coli Hsp70 homologue DnaK as full-length protein and its individual domains in the nucleotide-free and ATP-bound conformation. We found a surprising degree of flexibility in both domains. The comparison of the full-length protein with the isolated domains demonstrates a mutual stabilization of both domains. This protection from solvent was most pronounced and in addition was nucleotide-dependent in the lowerbeta-sheet of the SBD and the loop that connects the last beta-strand with helix alphaA. Interestingly, the linker region, which connects NBD and SBD and which is close to the protected loop in the SBD, is solvent-exposed in the absence of nucleotide and completely protected from hydrogen exchange in the presence of ATP. Peptide binding to DnaK.ATP reverts the ATP-induced conformational changes in the linker and selected parts of the NBD. Our data outline a pathway for allosteric interdomain control and suggest an important role of the linker and the base of helix alphaA.     

Allostery and Conformational Dynamics in cAMP-binding Acyltransferases

Mycobacteria harbor unique proteins that regulate protein lysine acylation in a cAMP-regulated manner. These lysine acyltransferases from Mycobacterium smegmatis (KATms) and Mycobacterium tuberculosis (KATmt) show distinctive biochemical properties in terms of cAMP binding affinity to the N-terminal cyclic nucleotide binding domain and allosteric activation of the C-terminal acyltransferase domain. Here we provide evidence for structural features in KATms that account for high affinity cAMP binding and elevated acyltransferase activity in the absence of cAMP. Structure-guided mutational analysis converted KATms from a cAMP-regulated to a cAMP-dependent acyltransferase and identified a unique asparagine residue in the acyltransferase domain of KATms that assists in the enzymatic reaction in the absence of a highly conserved glutamate residue seen in Gcn5-related N-acetyltransferase-like acyltransferases. Thus, we have identified mechanisms by which properties of similar proteins have diverged in two species of mycobacteria by modifications in amino acid sequence, which can dramatically alter the abundance of conformational states adopted by a protein.     

Structural dynamics in the activation of Epac

Epac1 is a cAMP-responsive exchange factor for the small G-protein Rap. It consists of a regulatory region containing a cyclic nucleotide binding (CNB) domain and a catalytic region that activates Rap. In the absence of cAMP, access of Rap to the catalytic site is blocked by the regulatory region. We analyzed the conformational states of the CNB domain in the absence and in the presence of cAMP and cAMP analogues by NMR spectroscopy, resulting in the first direct insights into the activation mechanism of Epac. We prove that the CNB domain exists in equilibrium between the inactive and the active conformation, which is shifted by binding of cAMP. cAMP binding results in conformational changes in both the ligand binding pocket and the outer helical segments. We used two different cAMP antagonists that block these successive changes to elucidate the steps of this process. Highlighting the role of dynamics, the superactivator 8-pCPT-2'-O-Me-cAMP induces similar conformational changes as cAMP but causes different internal mobility. The results reveal the critical elements of the CNB domain of Epac required for activation and highlight the role of dynamics in this process.     

Active Site Coupling in PDE:PKA Complexes Promotes Resetting of Mammalian cAMP Signaling

Cyclic 3′5′ adenosine monophosphate (cAMP)-dependent-protein kinase (PKA) signaling is a fundamental regulatory pathway for mediating cellular responses to hormonal stimuli. The pathway is activated by high-affinity association of cAMP with the regulatory subunit of PKA and signal termination is achieved upon cAMP dissociation from PKA. Although steps in the activation phase are well understood, little is known on how signal termination/resetting occurs. Due to the high affinity of cAMP to PKA (K_D ∼ low nM), bound cAMP does not readily dissociate from PKA, thus begging the question of how tightly bound cAMP is released from PKA to reset its signaling state to respond to subsequent stimuli. It has been recently shown that phosphodiesterases (PDEs) can catalyze dissociation of bound cAMP and thereby play an active role in cAMP signal desensitization/termination. This is achieved through direct interactions with the regulatory subunit of PKA, thereby facilitating cAMP dissociation and hydrolysis. In this study, we have mapped direct interactions between a specific cyclic nucleotide phosphodiesterase (PDE8A) and a PKA regulatory subunit (RIα isoform) in mammalian cAMP signaling, by a combination of amide hydrogen/deuterium exchange mass spectrometry, peptide array, and computational docking. The interaction interface of the PDE8A:RIα complex, probed by peptide array and hydrogen/deuterium exchange mass spectrometry, brings together regions spanning the phosphodiesterase active site and cAMP-binding sites of RIα. Computational docking combined with amide hydrogen/deuterium exchange mass spectrometry provided a model for parallel dissociation of bound cAMP from the two tandem cAMP-binding domains of RIα. Active site coupling suggests a role for substrate channeling in the PDE-dependent dissociation and hydrolysis of cAMP bound to PKA. This is the first instance, to our knowledge, of PDEs directly interacting with a cAMP-receptor protein in a mammalian system, and highlights an entirely new class of binding partners for RIα. This study also highlights applications of structural mass spectrometry combined with computational docking for mapping dynamics in transient signaling protein complexes. Together, these results present a novel and critical role for phosphodiesterases in moderating local concentrations of cAMP in microdomains and signal resetting.     

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.     

Structure and regulation of the cAMP-binding domains of Epac2

Cyclic adenosine monophosphate (cAMP) is a universal second messenger that, in eukaryotes, was believed to act only on cAMP-dependent protein kinase A (PKA) and cyclic nucleotide-regulated ion channels. Recently, guanine nucleotide exchange factors specific for the small GTP-binding proteins Rap1 and Rap2 (Epacs) were described, which are also activated directly by cAMP. Here, we have determined the three-dimensional structure of the regulatory domain of Epac2, which consists of two cyclic nucleotide monophosphate (cNMP)-binding domains and one DEP (Dishevelled, Egl, Pleckstrin) domain. This is the first structure of a cNMP-binding domain in the absence of ligand, and comparison with previous structures, sequence alignment and biochemical experiments allow us to delineate a mechanism for cyclic nucleotide-mediated conformational change and activation that is most likely conserved for all cNMP-regulated proteins. We identify a hinge region that couples cAMP binding to a conformational change of the C-terminal regions. Mutations in the hinge of Epac can uncouple cAMP binding from its exchange activity.     

Active Site Coupling in PDE:PKA Complexes Promotes Resetting of Mammalian cAMP Signaling

Cyclic 3′5′ adenosine monophosphate (cAMP)-dependent-protein kinase (PKA) signaling is a fundamental regulatory pathway for mediating cellular responses to hormonal stimuli. The pathway is activated by high-affinity association of cAMP with the regulatory subunit of PKA and signal termination is achieved upon cAMP dissociation from PKA. Although steps in the activation phase are well understood, little is known on how signal termination/resetting occurs. Due to the high affinity of cAMP to PKA (K_D ∼ low nM), bound cAMP does not readily dissociate from PKA, thus begging the question of how tightly bound cAMP is released from PKA to reset its signaling state to respond to subsequent stimuli. It has been recently shown that phosphodiesterases (PDEs) can catalyze dissociation of bound cAMP and thereby play an active role in cAMP signal desensitization/termination. This is achieved through direct interactions with the regulatory subunit of PKA, thereby facilitating cAMP dissociation and hydrolysis. In this study, we have mapped direct interactions between a specific cyclic nucleotide phosphodiesterase (PDE8A) and a PKA regulatory subunit (RIα isoform) in mammalian cAMP signaling, by a combination of amide hydrogen/deuterium exchange mass spectrometry, peptide array, and computational docking. The interaction interface of the PDE8A:RIα complex, probed by peptide array and hydrogen/deuterium exchange mass spectrometry, brings together regions spanning the phosphodiesterase active site and cAMP-binding sites of RIα. Computational docking combined with amide hydrogen/deuterium exchange mass spectrometry provided a model for parallel dissociation of bound cAMP from the two tandem cAMP-binding domains of RIα. Active site coupling suggests a role for substrate channeling in the PDE-dependent dissociation and hydrolysis of cAMP bound to PKA. This is the first instance, to our knowledge, of PDEs directly interacting with a cAMP-receptor protein in a mammalian system, and highlights an entirely new class of binding partners for RIα. This study also highlights applications of structural mass spectrometry combined with computational docking for mapping dynamics in transient signaling protein complexes. Together, these results present a novel and critical role for phosphodiesterases in moderating local concentrations of cAMP in microdomains and signal resetting.     

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.     

RIalpha subunit of PKA: a cAMP-free structure reveals a hydrophobic capping mechanism for docking cAMP into site B

In eukaryotes the primary target for cAMP, a ubiquitous second messenger, is cAMP-dependent protein kinase (PKA). Understanding how binding and release of cAMP changes the cAMP binding domains and then triggers long-range allosteric responses is an important challenge. This conformational switching requires structure solutions of cAMP binding domains in cAMP-bound and cAMP-free states. We describe for the first time a crystal structure of the cAMP binding domains of PKA type Ialpha regulatory subunit where site A is occupied by cGMP and site B is unoccupied. The structure reveals that the carboxyl terminus of domain B serves as a hydrophobic cap, locking the cyclic nucleotide via its adenine ring into the beta-barrel. In the absence of cAMP, the "cap" is released via an extension of the C-terminal helix. This simple hinge mechanism for binding and release of cAMP also provides a mechanism for allosteric communication between sites A and B.     

A generalized allosteric mechanism for cis-regulated cyclic nucleotide binding domains

Cyclic nucleotides (cAMP and cGMP) regulate multiple intracellular processes and are thus of a great general interest for molecular and structural biologists. To study the allosteric mechanism of different cyclic nucleotide binding (CNB) domains, we compared cAMP-bound and cAMP-free structures (PKA, Epac, and two ionic channels) using a new bioinformatics method: local spatial pattern alignment. Our analysis highlights four major conserved structural motifs: 1) the phosphate binding cassette (PBC), which binds the cAMP ribose-phosphate, 2) the “hinge,” a flexible helix, which contacts the PBC, 3) the β_2,3 loop, which provides precise positioning of an invariant arginine from the PBC, and 4) a conserved structural element consisting of an N-terminal helix, an eight residue loop and the A-helix (N3A-motif). The PBC and the hinge were included in the previously reported allosteric model, whereas the definition of the β_2,3 loop and the N3A-motif as conserved elements is novel. The N3A-motif is found in all cis-regulated CNB domains, and we present a model for an allosteric mechanism in these domains. Catabolite gene activator protein (CAP) represents a trans-regulated CNB domain family: it does not contain the N3A-motif, and its long range allosteric interactions are substantially different from the cis-regulated CNB domains.     

Ligand-induced conformational and structural dynamics changes in Escherichia coli cyclic AMP receptor protein

Cyclic AMP receptor protein (CRP) regulates the expression of a large number of genes in E. coli. It is activated by cAMP binding, which leads to some yet undefined conformational changes. These changes do not involve significant redistribution of secondary structures. A potential mechanism of activation is a ligand-induced change in structural dynamics. Hence, the cAMP-mediated conformational and structural dynamics changes in the wild-type CRP were investigated using hydrogen-deuterium exchange and Fourier transform infrared spectroscopy. Upon cAMP binding, the two functional domains within the wild-type CRP undergo conformational and structural dynamics changes in two opposite directions. While the smaller DNA-binding domain becomes more flexible, the larger cAMP-binding domain shifts to a less dynamic conformation, evidenced by a faster and a slower amide H-D exchange, respectively. To a lesser extent, binding of cGMP, a nonfunctional analogue of cAMP, also stabilizes the cAMP-binding domain, but it fails to mimic the relaxation effect of cAMP on the DNA-binding domain. Despite changes in the conformation and structural dynamics, cAMP binding does not alter significantly the secondary structural composition of the wild-type CRP. The apparent difference between functional and nonfunctional analogues of cAMP is the ability of cAMP to effect an increase in the dynamic motions of the DNA binding domain.