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Originally published In Press as doi:10.1074/jbc.M706231200 on September 4, 2007

J. Biol. Chem., Vol. 282, Issue 44, 32256-32263, November 2, 2007
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Conformational Analysis of Epac Activation Using Amide Hydrogen/Deuterium Exchange Mass Spectrometry*

Melissa Brock{ddagger}1, Fenghui Fan§1, Fang C. Mei§, Sheng Li{ddagger}, Christopher Gessner{ddagger}, Virgil L. Woods, Jr., Supported by NCI, National Institutes of Health, Public Health Service Grants CA099835, CA118595, GM037684, AI0220221, and AI022160 and Discovery Grant (UC10591) from the University of California Industry–University Cooperative Research Program{ddagger}2, and Xiaodong Cheng, Supported by NIGMS, National Institutes of Health, Public Health Service Grant GM066170 and American Heart Association Grant-in-Aid 0755049Y§3

From the §Department of Pharmacology and Toxicology and Sealy Center for Structural Biology and Molecular Biophysics, University of Texas Medical Branch, Galveston, Texas 77555-1031 and the {ddagger}Department of Medicine and Biomedical Sciences Graduate Program, University of California, San Diego, La Jolla, California 92093-0656

Received for publication, July 30, 2007 , and in revised form, September 4, 2007.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Exchange proteins directly activated by cAMP (Epac) play important roles in mediating the effects of cAMP through the activation of downstream small GTPases, Rap. To delineate the mechanism of Epac activation, we probed the conformation and structural dynamics of Epac using amide hydrogen/deuterium exchange and structural modeling. Our studies show that cAMP induces significant conformational changes that lead to a spatial rearrangement of the regulatory components of Epac and allows the exposure of the catalytic core for effector binding without imposing significant conformational change on the catalytic core. Homology modeling and comparative structural analyses of the cAMP binding domains of Epac and cAMP-dependent protein kinase (PKA) lead to a model of Epac activation, in which Epac and PKA activation by cAMP employs the same underlying principle, although the detailed structural and conformational changes associated with Epac and PKA activation are significantly different.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Exchange proteins directly activated by cAMP (Epac)4 are a family of novel guanine nucleotide exchange factors regulated by the second messenger cAMP (1, 2). Epac proteins have been shown to be involved in a myriad of cAMP-related cellular functions in parallel to the classic intracellular cAMP receptor, cAMP-dependent protein kinase (PKA) (3–12). There are two major isoforms of Epac, Epac1 and Epac2, each encoded by an independent gene and with distinctive tissue distribution in mammalian cells. Epac1 and Epac2 share extensive sequence homology, and both contain an N-terminal regulatory region and a C-terminal catalytic region. The catalytic region of Epac consists of a RAS exchange motif (REM) domain, RAS association (RA) domain, and a classic CDC25 homology domain (CDC25HD) responsible for nucleotide exchange activity. Whereas the regulatory region of Epac1 and Epac2 shares a Dishevelled/Egl-10/pleckstrin (DEP) domain followed by a cAMP-binding domain (CBD), an additional CBD N-terminal to the DEP domain is presented in Epac2. The function of this extra CBD domain (CBD-A) is not clear, since it binds cAMP with low affinity and does not seem to be essential for Epac regulation by cAMP (13).

Biochemical and structural analyses by Bos and Wittinghofer's groups have revealed a general scheme of Epac regulation (13–16). In the absence of cAMP, the N-terminal regulatory region of Epac acts as an autoinhibitor of the C-terminal guanine nucleotide exchange factor activity by sterically blocking the access of the downstream effector, Rap. Binding of cAMP to the high affinity CBD leads to a conformational change that releases the CDC25HD from the interdomain inhibition imposed by the CBD. This regulatory scheme is further supported by a recently solved crystal structure of the full-length Epac2 in the absence of cAMP (17). In this autoinhibited Epac2 structure, the regulatory and catalytic regions form two distinctive structural lobes. The smaller regulatory lobe is anchored to the larger catalytic lobe by the so-called "switchboard" structure, which is a five-strand beta-sheet-like structure consisting of the C terminus of the second CBD of Epac2 (CBD-B), the N terminus of the REM domain, and a hairpin of the catalytic core. Although CBD-A does not interact with the catalytic region, CBD-B of Epac2, which is common to Epac1 and Epac2, is briefly connected to the catalytic guanine nucleotide exchange factor domain through an "ionic latch" (17). This loose interdomain interaction is the only direct contact point between the regulatory region and catalytic core of Epac. Because of its proximity to the Rap1-binding surface, this ionic latch is believed to be important for the inhibitory effect exerted by the CBD-B on the catalytic activity.

Determining the specific regions/residues of Epac that undergo conformational changes in response to cAMP binding is the next step in elucidating the mechanism of Epac activation. Hydrogen exchange techniques have been successfully employed to study protein conformational change and protein folding for more than 30 years (18, 19). The incorporation of pepsin proteolysis, HPLC separation, and mass spectrometry techniques have significantly increased the resolution and usefulness of amide hydrogen exchange study (20–23). The development of an automated hydrogen exchange-liquid chromatography-mass spectrometry-based technology, referred to as enhanced deuterium exchange-mass spectrometry (DXMS), has further significantly improved the throughput, comprehensiveness, and resolution (24). DXMS is now a mature platform for studying protein structure (25–29), protein dynamics (30–38), protein-ligand (39–42), and protein-protein interactions (43–48) at the submolecular level. Since the atomic structure of cAMP-bound Epac in its active state is not currently available, the molecular mechanism of Epac activation is not known. In this study, we investigated the conformational and dynamic states of Epac1 in the presence and absence of cAMP using DXMS and structural modeling to delineate the mechanism of Epac activation.


Figure 1
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  		FIGURE 1.
Pepsin digestion maps of {Delta}(1–148)Epac1. Shown is the peptide fragmentation pattern (indicated by the solid lines) of cAMP-free {Delta}(1–148)Epac1.

 

   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
Protein Expression and Purification—Recombinant {Delta}(1–148)Epac1 was expressed and purified as described previously (49). All proteins were at least 95% pure, as judged by SDS-PAGE.

Optimization of Pepsin Digestion Conditions for DXMS Analysis—DXMS analysis was performed as previously described (31, 32, 43, 44). Two major parameters optimized were the concentration of guanidine hydrochloride in the quenching buffer and the pump C flow rate over the pepsin column. A 5-µl stock solution of {Delta}(1–148)Epac1 (8 mg/ml) was diluted with 15 µl of water and then quenched with 30 µl of 0.8% formic acid containing various concentrations of guanidine hydrochloride (0–6.4 M). The quenched sample was immediately injected into the DXMS apparatus, and the system was started. The flow rate passing over the pepsin column was varied between 100 and 250 µl/min.

Hydrogen-Deuterium Exchange—Deuterated samples were prepared at 23 ± 1 °C by diluting 5 µl of {Delta}(1–148)Epac1 stock solution with 15 µl of deuterated buffer (20 mM Mops (pH 7.0), 50 mM NaCl, 1 mM dithiothreitol), followed by "on-exchange" incubation for varying times (10–3000 s) prior to quenching in 30 µl of 0.8% formic acid, 3.2 M guanidine hydrochloride at 0 °C (31, 32, 43, 44). These functionally deuterated samples were then subjected to DXMS analysis, along with control samples of nondeuterated and fully deuterated {Delta}(1–148)Epac1 (incubated in deuterated buffer overnight at room temperature). Corrections for back-exchange were made as previously described (39). Typical deuteron recovery, as determined by analysis of equilibrium-deuterated Epac samples, was 77%.

DXMS Analysis—A 20-µl H/D-exchanged {Delta}(1–148)Epac1 protein solution was quenched by the addition of 30 µl of 3.2 M guanidine hydrochloride in 0.8% formic acid (0 °C) as previously described (31, 32, 43, 44). The quenched solution was frozen on dry ice and held at -80 °C until thawed with the use of a cryogenic autosampler as previously described (31, 32, 43, 44). Thawed samples (50 µl) were then automatically and immediately passed over a column (66-µl bed volume) filled with porcine pepsin (Sigma) (immobilized on Poros 20 AL medium at 30 mg/ml following the manufacturer's instructions), at a flow rate of 100 µl/min, with contemporaneous collection of the resulting proteolytic products by a C18 column (Vydac). Subsequently, the C18 column was eluted with a linear gradient of 5–45% B over 30 min (solvent A was 0.05% trifluoroacetic acid in water, and solvent B was 80% (v/v) acetonitrile, 20% (v/v) water, 0.01% trifluoroacetic acid). Mass spectrometric analyses were carried out with a Thermo Finnigan LCQ mass spectrometer with capillary temperature at 200 °C (31, 32, 43, 44). Data acquisition was performed in either MS1 profile mode (for deuterium quantification) or data-dependent MS2 mode (for peptide identification).

Sequence Identification of Peptide Fragments—The SEQUEST software program (Thermo Finnigan Inc.) was used to identify the likely sequence of the parent peptide ions. Identified peptides were then further examined to determine if the quality of the measured isotopic envelope of peptides was sufficient to allow accurate measurement of the geometric centroid of isotopic envelopes on deuterated samples and then assayed for deuterium content in functionally deuterated samples employing specialized software as previously described (31, 32, 43, 44).

Structural Modeling of Epac1—The Epac1 structural model was initially generated using homology modeling software MODELLER (50). The recently solved Epac2 three-dimensional coordinates (Protein Data Bank code 2BYV) (17) was used as the template. A three-dimensional model of the cAMP-bound Epac1 CBD was created using the CBD-B of rat PKA RIIbeta crystal structure (Protein Data Bank code 1CX4 [PDB] ) as a template. The initial structural models were subjected to additional energy minimization and further refined using the molecular dynamics simulation package AMBER in the presence of a water box. The final Epac1 models were based on the average energy-minimized structure over the last 100 ps of the 5-ns molecular dynamics simulation.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
Pepsin Fragmentation of {Delta}(1–148)Epac1 and Peptide Identification—Pepsin digestion and HPLC separation conditions that produced {Delta}(1–148)Epac1 fragments of optimal size and distribution for exchange analysis were established prior to H/D exchange experiments. We used {Delta}(1–148)Epac1 in this study, since the published literature so far indicates that the function of the N-terminal 148 residues is mainly associated with cellular targeting of Epac1 in vivo (13, 51). In addition, extensive biophysical and biochemical characterization by Wittinghofer and co-workers (14) show that {Delta}(1–148)Epac1 retains all measurable biochemical functional properties, such as cAMP binding and Rap activation. Removal of the first 148 residues significantly improves Epac1 solubility and increases expression levels in Escherichia coli, whereas the full-length Epac1 protein cannot be expressed and purified in sufficient quantity (14, 49). Optimal pepsin digestion for {Delta}(1–148)Epac1 was obtained by diluting one part of the deuterated sample with one and a half parts of the quench solution (3.2 M guanidine hydrochloride in 0.8% formic acid). The quenched sample was then run over immobilized pepsin with a duration of 40 s. These conditions generated 111 peptides covering 76% of the {Delta}(1–148)Epac1 sequence (Fig. 1).

H/D Exchanges of {Delta}(1–148)Epac1 in the cAMP-free State—To explore the intrinsic protein dynamics and conformational flexibility of Epac, H/D exchange of {Delta}(1–148)Epac1 was monitored by DXMS. Fig. 2 shows one specific peptide fragment, Pro487–Arg492, prior and subsequent to dilution in D2O. Incorporation of deuterium is clearly evident from the increase in overall mass and complexity of the peptide mass peaks as a function of deuteration time. The deuteration level of cAMP-free {Delta}(1–148)Epac1 as a function of time is illustrated in Fig. 3A. Peptide fragments with a broad range of exchange speeds, varying from nearly fully exchanged at the shortest time point employed (10 s) to no detectable exchange at the longest time point employed (3000 s), were observed.


Figure 2
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  		FIGURE 2.
Mass spectra of pepsin-digested {Delta}(1–148)Epac1 with and without deuteration. A, initial isotopic envelope for peptide fragment Pro486–Arg492 before deuteration. B, final isotopic envelope for peptide fragment Pro486–Arg492 after deuteration showing increased backbone amide deuteron incorporation as indicated by the shifting of the isotopic envelope to a higher m/z ratio.

 
When we mapped the solvent-exposed peptides, defined as more than 50% deuteration after 100 s in D2O, onto the homology structural model of Epac1 constructed based on the x-ray structure of Epac2, many of these fragments were located in the loop and turn regions (Fig. 3B). This is consistent with the fact that loops and turns are usually exposed to solvent and more flexible. In fact, the fastest exchange fragments revealed by DXMS correspond to regions in Epac2 structure without significant electron density, a sign of high flexibility associated with structure disorder. Most of the fast exchange regions of {Delta}(1–148)Epac1 are located in the CBD, REM, and RA domains, suggesting that these domains, particularly the CBD, are highly dynamic in the absence of cAMP. On the other hand, the catalytic core of Epac (CDC25HD), except for the very C-terminal tail, is relatively solvent-inaccessible.


Figure 3
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  		FIGURE 3.
Amide H/D exchange results of cAMP-free {Delta}(1–148)Epac1. A, the deuteration levels of each peptide fragment at various time points are shown by different colors, from blue (<10%) to red (>90%), as indicated at the lower right corner of the figure. Each block represents an individual peptide analyzed at each of the six time points (from top, 10, 30, 100, 300, 1000, and 3000 s). B, peptide fragments with more than 50% deuteration levels after a 100-s exchange in D2O are mapped onto the structural model of Epac1 in red (>70%) and in yellow (>50% and <70%). Three independent DXMS analyses were performed, and similar results were obtained. Studies performed with our automated exchange data acquisition apparatus result in deuterium incorporation S.D. values of 2.5% or less when replicates are analyzed (33).

 
Effects of cAMP Binding on H/D Exchanges of {Delta}(1–148)Epac1—To elucidate the conformational changes associated with Epac activation, the time-dependent amide H/D exchange patterns of {Delta}(1–148)Epac1 in the presence and absence of cAMP were measured and compared. Although the binding of cAMP had no effect on the time-dependent incorporation of deuterons for most of the peptide fragments, as typified by peptide Phe379–Leu392 (Fig. 4A), the rates and/or the extents of the H/D exchange of several peptides decreased in response to cAMP binding over the experimental time course (Fig. 4, B–H), indicating that these peptide fragments are protected by the binding of cAMP. We did not observe that cAMP significantly increased the rate of deuteron incorporation for any of the peptide fragments analyzed.

When the peptide fragments of Epac1 that underwent significant changes in amide H/D exchange were mapped onto the three-dimensional structural model of Epac1, all changes in solvent accessibility induced by cAMP were located at the relative more dynamic parts of the Epac, including the CBD, the switchboard, and the REM domains. The catalytic core (CDC25HD) domain remains constant upon cAMP binding (Fig. 5A). It is reassuring that the largest changes induced by cAMP binding are located directly at the cAMP binding pocket. These apparent decreases in H/D exchange at the cAMP binding pocket upon cAMP binding may be due to the steric protection of residues that come in direct contact with the ligand or/and to local conformational/dynamic changes in response to binding of cAMP. Although our DXMS analyses could not distinguish these possibilities, the coincidence between the observed major region of solvent protection and the cAMP binding pocket provides an independent validation for our DXMS analysis. On the other hand, observed changes in solvent accessibility at regions outside the cAMP binding pocket provided a potential description of the conformational changes induced by cAMP binding during Epac activation (Fig. 5A).

Comparative Molecular Dynamics Analyses of Epac1 CBD in cAMP-free and cAMP-bound States—To help interpret the observed potential conformational changes revealed by DXMS analysis and to further investigate the role of the switchboard in Epac activation, we generated a cAMP-bound Epac1 CBD homology model using the CBD-B of PKA-RIIbeta crystal structure as a template. Energy-minimized structural models of cAMP-free and cAMP-bound CBD of Epac1, based on crystal structures of cAMP-free Epac2 and cAMP-bound PKA-RIIbeta, respectively, were subjected to molecular dynamics simulations. Stable cAMP-free and cAMP-bound Epac1 CBD structures were obtained. Superposition of the cAMP-free and ligand-bound CBD models of Epac1 suggests that although the CBD is more solvent-accessible in the ligand-free state, as revealed by DXMS analysis in this study, the overall conformation of the beta-barrel core is similar between the cAMP-free state and ligand-bound state. The most significant conformational changes in the CBD of Epac1 between the cAMP-free state and cAMP-bound state appear to localize at the C-terminal helix/lid region (Fig. 5B), particularly the first two beta-strands of the switchboard. In the absence of the ligand, this segment, composed of residues 298–325, is in an extended conformation, pointing away from the beta-barrel core. In the presence of cAMP, the C-helix/switchboard undergoes a large hinge motion around Phe300 and folds back toward the beta-barrel core to cap the ligand like a "lid" into the cAMP-binding pocket. This hinge-capping mechanism for binding of cAMP has been demonstrated in PKA by comparing the crystal structures of the CBD of RI subunit in the cAMP-free state and ligand-bound state (52) and supported by our DXMS data, since binding of cAMP leads to significant protection of the switchboard.


Figure 4
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  		FIGURE 4.
Deuterium incorporation changes in {Delta}(1–148)Epac1 upon binding of cAMP. Time-dependent deuterium incorporation in Epac peptide fragments Phe379–Leu392 (A), Phe232–Trp241 (B), Val251–Asp267 (C), Ala272–Pro278 (D), Glu315–Leu322 (E), Ser348–Leu357 (F), Leu438–Phe447 (G), and Leu448–Leu454 (H). Data shown in closed and open circles represent deuterium incorporation into apo- and cAMP-bound {Delta}(1–148)Epac at various time intervals after being dissolved in D2O at room temperature.

 
As shown in Fig. 5B, the conserved Leu273 (orange) of the phosphate binding cassette (PBC) in the absence of cAMP occupies the same space as the conserved Phe300 (green) at the hinge region in the cAMP-bound structure. The bending of the C-helix is only possible in the presence of cAMP, which repositions the PBC and releases steric hindrance imposed by Leu273. In addition, the cAMP-bound Epac CBD model also revealed several important interactions that anchored and stabilized the lid structure in the presence of cAMP. These interactions include a hydrogen bonding network formed between Asp267 and Lys310, Gln270 and Thr311, and Asn275 and Glu315 (Fig. 6A); ionic interactions between Asp267 and Lys310 and between Lys256 and Glu325 (Fig. 6B); and hydrophobic interactions formed among Leu273, Phe300, and Ile304 at the hinge regions as well as among Leu271, Leu314, and the adenine of cAMP (Fig. 6C). Since all of these residues are conserved among Epac1 and the second high affinity cAMP-binding domain of Epac2, these interactions are probably functionally important for Epac activation.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
The cAMP-binding domain, a conserved structural module, acts as a common molecular switch for controlling the biological activities of both eukaryotic intracellular cAMP receptors, PKA and Epac. Although the crystal structures of the cAMP-bound regulatory subunit and a partial PKA holoenzyme complex, along with extensive biochemical and structural analyses, have revealed a clear picture regarding the interactions between CBD and the catalytic subunit and the detailed mechanism of PKA activation by cAMP (53, 54), the mechanism of Epac activation by cAMP is not all clear. In the PKA holoenzyme complex of RI{alpha}-(91–244)·C, the CBD-A of RI{alpha} (91–244) makes extensive contact with the C-subunit. The B- and C-helices of CBD-A snap into a single extended helix that is anchored and stabilized by the catalytic subunit and, in particular, the activation loop. The PBC is stretched away from the beta-barrel to interact directly with the G-helix of the catalytic subunit in the complex (54). Activation of PKA holoenzyme, initiated by the binding of cAMP to the R-subunit, is accompanied by widespread conformational changes of the R-subunit, demonstrated extensively by x-ray crystallographic analyses (53, 54) and biophysical studies in solution (55). Binding of the cAMP results in the retraction of the PBC in the direction of the cAMP binding pocket and global reorientation of the subhelical domain of CBD-A. The pivot motion around the hydrophobic hinge dislodges the single extended B/C-helix, and subsequently the inhibitor sequence, from the docking site on the C-subunit. In the absence of stabilization/anchoring effects of the C-subunit, the B/C-helix bends in the middle to form two individual helices with the C-helix portion folded back onto the beta-barrel to form the "lid" of the cAMP-binding pocket. These extensive cAMP-induced conformation changes eventually cause the dissociation of the PKA holoenzyme.

Although it is likely that Epac and PKA activations share the same underlying principle, the detailed mechanisms most likely will be significantly different, since Epac and PKA share little sequence and structural similarity outside of the CBD domain. This notion is supported by the recently published cAMP-free Epac2 structure, which reveals that the corresponding lid in Epac actually forms the first beta-strand of the switchboard instead of a helix as in PKA, despite the fact that the lid region of Epac and PKA share extensive sequence homology. In addition, there is only one brief direct contact point between the CBD and catalytic core of Epac, described as the "ionic latch" in its autoinhibited state. This is in startling contrast to the extensive interactions observed between the CBD of the regulatory subunit and the catalytic subunit in PKA holoenzyme.

Since the structure of cAMP-bound Epac in its active state is not currently available, we applied hydrogen/deuterium exchange analysis to investigate the conformational and dynamic changes associated with Epac activation. Amide H/D exchange has been used extensively to analyze structural dynamics and conformational changes in proteins, since the rate of an amide proton with the solvent is largely determined by the flexibility and motion around the exchanging proton. Our DXMS studies reveal some important findings that are not provided by the cAMP-free Epac2 crystal structure. Overall, Epac is more dynamic in the absence of cAMP, particularly for the regulatory region, including the CBD and the switchboard region. On the other hand, the catalytic core (CDC25HD) is relatively rigid. Although parts of the REM and RA domains are also relatively flexible, the switchboard region is one of the most dynamic parts of the Epac molecule. Hinge prediction based on gaussian network model first normal model displacement analysis also predicts this region as the major hinge for conformational fluctuations (56). The CBD, switchboard, and parts of the REM domain become more protected in the presence of cAMP, whereas the conformation of CDC25HD catalytic core does not change significantly with or without cAMP. Taken together, these results suggest that binding of cAMP induces significant conformational changes that center on the switchboard segment, which consequently leads to a spatial rearrangement of the regulatory components of Epac and allows for the exposure of the catalytic core for effector binding without imposing significant conformational change on the catalytic core.


Figure 5
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  		FIGURE 5.
Conformational changes induced by cAMP as revealed by DXMS. A, peptide fragments protected by cAMP are mapped to the ribbon diagram of Epac1 in purple. The DEP, CBD, REM, RA, and CDC25HD domains are colored in pink, green, blue, red, and cyan, respectively. B, structure of Epac1 CBD in the presence (green) and absence (orange) of cAMP based on homology modeling and molecular dynamics simulations. Locations of the C-helix, hinge, PBC, and residues Leu273 and Phe300 are indicated by arrows.

 


Figure 6
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  		FIGURE 6.
Proposed interactions important for stabilizing the lid of cAMP-binding pocket in cAMP-bound Epac. A, hydrogen bonding network formed between Asp267 and Lys310, Gln270 and Thr311, and Asn275 and Glu315. B, ionic interactions between Asp267 and Lys310 and between Lys256 and Glu325. C, hydrophobic interactions formed among Leu273, Phe300, and Ile304 at the hydrophobic hinge region and among Leu271, Leu314, and the adenine ring of cAMP.

 


Figure 7
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  		FIGURE 7.
Mechanism of Epac activation. Schematic representation of cAMP-induced Epac activation with the DEP, CBD, REM, RA, and CDC25HD domains colored in pink, green, blue, red, and cyan, respectively. In the cAMP-free, inactive Epac state, the regulatory region is anchored by the switchboard to keep the CBD in close proximity to the catalytic core to prevent the binding of Rap. Upon binding of cAMP, the beta-strands 1 and 2 (S1 and S2) break away from the switchboard, fold back as an {alpha}-helix toward the beta-barrel, and interact directly with cAMP to form the base of the cAMP-binding pocket. This localized hinge motion coupled with a switch of the secondary structure of the lid reorients the CDC25HD relative to CBD and ultimately leads to the exposure of the catalytic core of Epac for the access of Rap GTPase. This model is based on a previously published model as proposed by Rehmann et al. (17).

 
Earlier biochemical and structural studies by the Bos and Wittinghofer groups suggest that the LID region of the C terminus of CBD in Epac plays an important role in the communication between the regulatory and catalytic domains and is pivotal for the activation of Epac by cAMP as in the case of PKA (13, 15, 16). It was proposed that regulation of Epac is dictated by the equilibrium between an inactive and an active state. In the absence of cAMP, Epac exists mostly in the inactive states, in which the LID region, particularly the conserved VLVLE motif, interacts with the catalytic region and prevents interaction between Epac and Rap. Binding of cAMP to Epac leads to a conformational change that allows the LID/VLVLE motif to move away from the catalytic core and closer to the cAMP binding pocket to interact directly with the cAMP and consequently releases the inhibitory effect of the VLVLE and permits the binding of Rap (15). However, the recently solved crystal structure of the full-length Epac2 in its inactive, ligand-free state reveals that the LID/VLVLE motif is not in the form of a helix as in the case of PKA; nor does the LID/VLVLE motif interact directly with the catalytic core as originally proposed. Instead, the Epac LID segment is part of a five-strand, beta-sheet-like "switchboard" structure (17). In light of our DXMS results and this substantial divergence of the lid structure between Epac and PKA, we performed an in-depth comparative sequence and structure analyses of Epac and PKA. Our study leads to a model of Epac activation (Fig. 7). In this model, the regulatory and the catalytic regions of Epac are held together by the switchboard to keep the CBD in close proximity of the catalytic core in order to prevent the binding of Rap. The C-helix and LID/VLVLE motif are anchored and stabilized by the N terminus of REM in a three-strand beta-sheet structure as part of the switchboard, leaving the CBD in an open and flexible conformation. Conformational changes induced by cAMP during Epac activation are centered on the switchboard, including parts of the REM domain, whereas the catalytic core of Epac remains relatively unchanged. Binding of cAMP drags the highly conserved PBC in the direction of the cAMP and reorients the C-helix, tethered to the PBC by a hydrophobic hinge, toward the beta-barrel of the CBD. This hinge movement pulls beta-strands 1 and 2 away from the five-strand beta-sheet of the switchboard. Breaking away of beta-strands 1 and 2 from the rest of the beta-sheet enables them to switch to an {alpha}-helix, which interacts directly with cAMP to form the lid of the cAMP-binding pocket. The conformational changes induced upon cAMP binding result in a closed CBD conformation and reorientation of the CBD/DEP domains relative to the rest of the molecule, which releases the catalytic core from the inhibitory contact imposed by the CBD. This model is based on a previous model proposed by Rehmann et al. (17) and supported by our DXMS results and structural analysis. Our earlier studies using Epac-based fluorescence resonance energy transfer indicators suggest that binding of cAMP leads to a more extended Epac conformation (57), an observation in agreement with our model. In addition, it has been shown that replacing the invariant leucine (Leu273) in the conserved PBC motif with a bulky tryptophan that blocks the hinge swinging and stabilizes the open conformation results in an inactive Epac molecule even in the presence of a saturating concentration of cAMP, supporting the significance of the hinge motion in Epac activation (16). Furthermore, secondary structure predictions based on several of the most commonly used algorithms all predict the lid of Epac as {alpha}-helical structure, instead of beta-strands. In fact, the corresponding regions of all known CBD proteins, including the cAMP-receptor protein, PKA, and cyclic-nucleotide-regulated ion channels, all exist as helical structures (53, 54, 58, 59). Therefore, it is conceivable that during Epac activation, because of the helical propensity, the first two beta-strands of the switchboard switch conformation from a beta-strand structure to form an extension of the C-helix to cap the cAMP-binding pocket.


   FOOTNOTES
 
* The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

1 Both contributed equally to this work. Back

2 To whom correspondence may be addressed: Dept. of Medicine, University of California, San Diego, 9500 Gilman Dr., La Jolla CA 92093-0656. Tel.: 858-534-2180; Fax: 858-534-2606; E-mail: vwoods{at}ucsd.edu. 3 To whom correspondence may be addressed: Dept. of Pharmacology and Toxicology, University of Texas Medical Branch, 301 University Blvd., Galveston, TX 77555-1031. Tel.: 409-772-9656; Fax: 409-772-9642; E-mail: xcheng{at}utmb.edu.

4 The abbreviations used are: Epac, exchange protein(s) directly activated by cAMP; CBD, cAMP binding domain; DXMS, deuterium exchange mass spectrometry; DEP, Dishevelled, Egl-10, pleckstrin; H/D, hydrogen/deuterium; PBC, phosphate binding cassette; PKA, cAMP-dependent protein kinase; C-subunit, catalytic subunit of PKA; R-subunit, regulatory subunit of PKA; RA, Ras association domain; Rap, Ras-proximate; REM, Ras exchange motif; CDC25HD, CDC25 homology domain; HPLC, high pressure liquid chromatography; Mops, 4-morpholinepropanesulfonic acid. Back



   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 

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