The Experts below are selected from a list of 6999 Experts worldwide ranked by ideXlab platform
William N. Zagotta - One of the best experts on this subject based on the ideXlab platform.
-
structure and energetics of allosteric regulation of hcn2 ion channels by Cyclic nucleotides
Journal of Biological Chemistry, 2016Co-Authors: Hannah A. Deberg, William N. Zagotta, Galen E. Flynn, Peter S. Brzovic, Stefan StollAbstract:Hyperpolarization-activated Cyclic nucleotide-gated (HCN) ion channels play an important role in regulating electrical activity in the heart and brain. They are gated by the binding of Cyclic nucleotides to a conserved, intracellular Cyclic Nucleotide-Binding Domain (CNBD), which is connected to the channel pore by a C-linker region. Binding of Cyclic nucleotides increases the rate and extent of channel activation and shifts it to less hyperpolarized voltages. We probed the allosteric mechanism of different Cyclic nucleotides on the CNBD and on channel gating. Electrophysiology experiments showed that cAMP, cGMP, and cCMP were effective agonists of the channel and produced similar increases in the extent of channel activation. In contrast, electron paramagnetic resonance (EPR) and nuclear magnetic resonance (NMR) on the isolated CNBD indicated that the induced conformational changes and the degrees of stabilization of the active conformation differed for the three Cyclic nucleotides. We explain these results with a model where different allosteric mechanisms in the CNBD all converge to have the same effect on the C-linker and render all three Cyclic nucleotides similarly potent activators of the channel.
-
Structural Mechanism for the Regulation of HCN Ion Channels by the Accessory Protein TRIP8b
Structure (London England : 1993), 2015Co-Authors: Hannah A. Deberg, William N. Zagotta, John R. Bankston, Joel C. Rosenbaum, Peter S. Brzovic, Stefan StollAbstract:Hyperpolarization-activated Cyclic nucleotide-gated (HCN) ion channels underlie the cationic Ih current present in many neurons. The direct binding of Cyclic AMP to HCN channels increases the rate and extent of channel opening and results in a depolarizing shift in the voltage dependence of activation. TRIP8b is an accessory protein that regulates the cell surface expression and dendritic localization of HCN channels and reduces the Cyclic nucleotide dependence of these channels. Here, we use electron paramagnetic resonance (EPR) to show that TRIP8b binds to the apo state of the Cyclic nucleotide binding Domain (CNBD) of HCN2 channels without changing the overall Domain structure. With EPR and nuclear magnetic resonance, we locate TRIP8b relative to the HCN channel and identify the binding interface on the CNBD. These data provide a structural framework for understanding how TRIP8b regulates the Cyclic nucleotide dependence of HCN channels.
-
Allosteric Regulation of the Cyclic Nucleotide-Binding Domain in HCN Channels
Biophysical Journal, 2015Co-Authors: Hannah A. Deberg, Shahidul M. Islam, Michael C. Puljung, Benoît Roux, William N. Zagotta, Stefan StollAbstract:Hyperpolarization-activated Cyclic nucleotide-gated (HCN) ion channels play an important role in regulating pacemaking activity in the heart and brain. They are regulated by the binding of Cyclic nucleotides to a conserved, intracellular Cyclic Nucleotide-Binding Domain (CNBD). Binding of Cyclic nucleotides increases the rate and extent of activation of the channels and shifts channel activation to less hyperpolarized voltages. In intact channels, cAMP and cGMP are full agonists and cCMP is a partial agonist. We use double electron-electron resonance (DEER) to study the conformational change associated with the binding of these three different Cyclic nucleotide species to the CNBD. We find that conformational changes associated with ligand binding vary depending on the Cyclic nucleotide bound and the location in the CNBD. We use the restrained ensemble MD (re-MD) simulations method to generate structural models integrating the complete set of experimentally measured data from DEER distance distribution histograms that describe the separation between pairs of spin labels attached to the CNBD. Our results indicate that, in the B-helix, binding of cGMP or cCMP has an effect intermediate to that of cAMP. However, in the C-helix cAMP and cCMP exhibit similar effects, but cGMP produces an intermediate effect between the apo and bound cAMP/cCMP DEER distance distributions. These data provide an interesting lens for studying allostery in HCN channels and indicate that the mechanism of protein allostery in the CNBD varies for different Cyclic nucleotides.
-
Flavonoid regulation of HCN2 channels.
The Journal of biological chemistry, 2013Co-Authors: Anne E. Carlson, Joel C. Rosenbaum, Tinatin I. Brelidze, Rachel E. Klevit, William N. ZagottaAbstract:The hyperpolarization-activated Cyclic nucleotide-modulated (HCN) channels are pacemaker channels whose currents contribute to rhythmic activity in the heart and brain. HCN channels open in response to hyperpolarizing voltages, and the binding of cAMP to their Cyclic Nucleotide-Binding Domain (CNBD) facilitates channel opening. Here, we report that, like cAMP, the flavonoid fisetin potentiates HCN2 channel gating. Fisetin sped HCN2 activation and shifted the conductance-voltage relationship to more depolarizing potentials with a half-maximal effective concentration (EC50) of 1.8 μM. When applied together, fisetin and cAMP regulated HCN2 gating in a nonadditive fashion. Fisetin did not potentiate HCN2 channels lacking their CNBD, and two independent fluorescence-based binding assays reported that fisetin bound to the purified CNBD. These data suggest that the CNBD mediates the fisetin potentiation of HCN2 channels. Moreover, binding assays suggest that fisetin and cAMP partially compete for binding to the CNBD. NMR experiments demonstrated that fisetin binds within the cAMP-binding pocket, interacting with some of the same residues as cAMP. Together, these data indicate that fisetin is a partial agonist for HCN2 channels.
-
Dimeric TRIP8b Binds to the Cyclic Nucleotide Binding Domain of HCN Channels
Biophysical Journal, 2013Co-Authors: John R. Bankston, Stacey S. Camp, William N. ZagottaAbstract:The accessory protein TRIP8b has been shown to modulate expression level, cellular localization, and Cyclic nucleotide-dependent regulation of hyperpolarization-activated Cyclic nucleotide-gated (HCN) channels. The mechanism through which TRIP8b exerts these effects is poorly understood. Work from this lab, as well as others, has shown that TRIP8b and HCN2 interact in two places. The first site is between the terminal three residues of HCN and the tetratricopeptide repeat (TPR) Domains of TRIP8b. We previously solved the X-ray crystal structure of this binding site bound to a peptide of the terminal 7 amino acids of HCN2. The second interaction involves the Cyclic Nucleotide-Binding Domain (CNBD) of HCN and a highly conserved region of TRIP8b N-terminal to the TPR Domains. This binding site is sufficient to impact the Cyclic nucleotide dependence of HCN channels. Here we use biochemical and biophysical techniques on isolated Domains of each protein to begin to understand the second interaction site between the CNBD and the TRIP8b conserved Domain. Light-scattering size-exclusion chromatography demonstrated that TRIP8b and HCN2 form a 2:1 complex when co-expressed and that TRIP8b, when expressed alone, forms both dimer and monomer. Surprisingly the CNBD of HCN2 interacts preferentially with the dimeric form of TRIP8b. In contrast, using a fluorescently labeled peptide of the terminal 7 amino acids of HCN2, we demonstrate that the carboxy terminal tripeptide of HCN2 is able to bind both the dimeric and monomeric forms of TRIP8b. The ability of the CNBD to discriminate between monomeric and dimeric TRIP8b suggests an interesting potential mechanism through which the impact of binding on the Cyclic nucleotide-dependent regulation of HCN channels can be tuned by controlling the oligomeric state of TRIP8b.
Holger Rehmann - One of the best experts on this subject based on the ideXlab platform.
-
Structure of Epac2 in complex with a Cyclic AMP analogue and RAP1B
Nature, 2008Co-Authors: Holger Rehmann, Ernesto Arias-palomo, Michael A. Hadders, Frank Schwede, Oscar Llorca, Johannes L. BosAbstract:Epac proteins are activated by binding of the second messenger cAMP and then act as guanine nucleotide exchange factors for Rap proteins. The Epac proteins are involved in the regulation of cell adhesion and insulin secretion. Here we have determined the structure of Epac2 in complex with a cAMP analogue (Sp-cAMPS) and RAP1B by X-ray crystallography and single particle electron microscopy. The structure represents the cAMP activated state of the Epac2 protein with the RAP1B protein trapped in the course of the exchange reaction. Comparison with the inactive conformation reveals that cAMP binding causes conformational changes that allow the Cyclic nucleotide binding Domain to swing from a position blocking the Rap binding site towards a docking site at the Ras exchange motif Domain.
-
Characterization of the Activation of the Rap‐Specific Exchange Factor Epac by Cyclic Nucleotides
Methods in Enzymology, 2006Co-Authors: Holger RehmannAbstract:Epac1 and Epac2 are cAMP‐dependent guanine nucleotide exchange factors (GEF) for the small G‐proteins Rap1 and Rap2. Epac is inactive in the absence of cAMP, and binding of cAMP to a Cyclic nucleotide–binding Domain in the N‐terminal regulatory region results in activation of the protein. The cAMP‐dependent activity of Epac proteins can be analyzed by a fluorescence‐based assay in vitro. These kinds of measurements can help to unravel the molecular mechanism by which cAMP binding is translated in activation of the protein. For this purpose, Epac mutants can be analyzed. In addition, the interaction of cAMP itself might be the focus of the research. Thus, modified cAMP analogs can be characterized by their ability to activate Epac. This is of particular interest for the development of Epac‐specific analogs, which do not act on other cellular cAMP targets such as protein kinase A (PKA) or for the design of therapeutic agents targeting Epac.
-
Biochemistry of the Rap-specific guanine nucleotide exchange factors PDZ-GEF1 and -2.
Methods in enzymology, 2006Co-Authors: H. Bea Kuiperij, Holger Rehmann, Fried J. T. ZwartkruisAbstract:PDZ‐GEFs represent one of four types of highly conserved Rap‐specific guanine nucleotide exchange factors. They contain a number of well‐known protein Domains, including a “related to Cyclic nucleotide binding Domain” (RCBD), a PDZ‐Domain, a Ras‐associating Domain (RA), and, of course, a catalytic Domain required for their exchange activity. Since their cloning more than 5 years ago, relatively little has been learned about their mode of regulation. Although their activity may in part depend on regulated membrane localization by means of the RA and/or PDZ Domain, it seems highly likely that PDZ‐GEFs can be modified by additional mechanisms as well. Based on analogy of the regulatory mechanisms of the cAMP‐responsive GEF Epac, in the past we postulated a role for the RCBD Domain in this. In this chapter, we give a detailed description of the methods that were used to unravel this mechanism in vitro and in vivo.
-
Characterization of the activation of the Rap-specific exchange factor Epac by Cyclic nucleotides.
Methods in enzymology, 2006Co-Authors: Holger RehmannAbstract:Epac1 and Epac2 are cAMP‐dependent guanine nucleotide exchange factors (GEF) for the small G‐proteins Rap1 and Rap2. Epac is inactive in the absence of cAMP, and binding of cAMP to a Cyclic nucleotide–binding Domain in the N‐terminal regulatory region results in activation of the protein. The cAMP‐dependent activity of Epac proteins can be analyzed by a fluorescence‐based assay in vitro. These kinds of measurements can help to unravel the molecular mechanism by which cAMP binding is translated in activation of the protein. For this purpose, Epac mutants can be analyzed. In addition, the interaction of cAMP itself might be the focus of the research. Thus, modified cAMP analogs can be characterized by their ability to activate Epac. This is of particular interest for the development of Epac‐specific analogs, which do not act on other cellular cAMP targets such as protein kinase A (PKA) or for the design of therapeutic agents targeting Epac.
Dieter Willbold - One of the best experts on this subject based on the ideXlab platform.
-
resonance assignment of the ligand free Cyclic nucleotide binding Domain from the murine ion channel hcn2
Biomolecular Nmr Assignments, 2015Co-Authors: S Schunke, Matthias Stoldt, U B Kaupp, Justin Lecher, Claudia Borger, Friederike Winkhaus, Dieter WillboldAbstract:Hyperpolarization activated and Cyclic nucleotide-gated (HCN) ion channels as well as Cyclic nucleotide-gated (CNG) ion channels are essential for the regulation of cardiac cells, neuronal excitability, and signaling in sensory cells. Both classes are composed of four subunits. Each subunit comprises a transmembrane region, intracellular N- and C-termini, and a C-terminal Cyclic Nucleotide-Binding Domain (CNBD). Binding of Cyclic nucleotides to the CNBD promotes opening of both CNG and HCN channels. In case of CNG channels, binding of Cyclic nucleotides to the CNBD is sufficient to open the channel. In contrast, HCN channels open upon membrane hyperpolarization and their activity is modulated by binding of Cyclic nucleotides shifting the activation potential to more positive values. Although several high-resolution structures of CNBDs from HCN and CNG channels are available, the gating mechanism for murine HCN2 channel, which leads to the opening of the channel pore, is still poorly understood. As part of a structural investigation, here, we report the complete backbone and side chain resonance assignments of the murine HCN2 CNBD with part of the C-linker.
-
structural insights into conformational changes of a Cyclic nucleotide binding Domain in solution from mesorhizobium loti k1 channel
Proceedings of the National Academy of Sciences of the United States of America, 2011Co-Authors: S Schunke, Matthias Stoldt, U B Kaupp, Justin Lecher, Dieter WillboldAbstract:Cyclic nucleotide-sensitive ion channels, known as HCN and CNG channels, are activated by binding of ligands to a Domain (CNBD) located on the cytoplasmic side of the channel. The underlying mechanisms are not well understood. To elucidate the gating mechanism, structures of both the ligand-free and -bound CNBD are required. Several crystal structures of the CNBD from HCN2 and a bacterial CNG channel (MloK1) have been solved. However, for HCN2, the cAMP-free and -bound state did not reveal substantial structural rearrangements. For MloK1, structural information for the cAMP-free state has only been gained from mutant CNBDs. Moreover, in the crystal, the CNBD molecules form an interface between dimers, proposed to be important for allosteric channel gating. Here, we have determined the solution structure by NMR spectroscopy of the cAMP-free wild-type CNBD of MloK1. A comparison of the solution structure of cAMP-free and -bound states reveals large conformational rearrangement on ligand binding. The two structures provide insights on a unique set of conformational events that accompany gating within the ligand-binding site.
-
Resonance assignments of the nucleotide-free wildtype MloK1 Cyclic Nucleotide-Binding Domain
Biomolecular Nmr Assignments, 2010Co-Authors: S Schunke, U. Benjamin Kaupp, Matthias Stoldt, Justin Lecher, Dieter WillboldAbstract:Cyclic nucleotide-sensitive ion channels, known as HCN and CNG channels play crucial roles in neuronal excitability and signal transduction of sensory cells. These channels are activated by binding of Cyclic nucleotides to their intracellular Cyclic Nucleotide-Binding Domain (CNBD). A comparison of the structures of wildtype ligand-free and ligand-bound CNBD is essential to elucidate the mechanism underlying nucleotide-dependent activation of CNBDs. We recently reported the solution structure of the Mesorhizobium loti K1 (MloK1) channel CNBD in complex with cAMP. We have now extended these studies and achieved nearly complete assignments of 1H, 13C and 15N resonances of the nucleotide-free CNBD. A completely new assignment of the nucleotide-free wildtype CNBD was necessary due to the sizable chemical shift differences as compared to the cAMP bound CNBD and the slow exchange behaviour between both forms. Scattering of these chemical shift differences over the complete CNBD suggests that nucleotide binding induces significant overall conformational changes.
-
solution structure of the mesorhizobium loti k1 channel Cyclic nucleotide binding Domain in complex with camp
EMBO Reports, 2009Co-Authors: S Schunke, Matthias Stoldt, Kerstin Novak, U B Kaupp, Dieter WillboldAbstract:Cyclic nucleotide-sensitive ion channels, known as HCN and CNG channels, are crucial in neuronal excitability and signal transduction of sensory cells. HCN and CNG channels are activated by binding of Cyclic nucleotides to their intracellular Cyclic Nucleotide-Binding Domain (CNBD). However, the mechanism by which the binding of Cyclic nucleotides opens these channels is not well understood. Here, we report the solution structure of the isolated CNBD of a Cyclic nucleotide-sensitive K+ channel from Mesorhizobium loti. The protein consists of a wide anti-parallel β-roll topped by a helical bundle comprising five α-helices and a short 310-helix. In contrast to the dimeric arrangement (‘dimer-of-dimers') in the crystal structure, the solution structure clearly shows a monomeric fold. The monomeric structure of the CNBD supports the hypothesis that the CNBDs transmit the binding signal to the channel pore independently of each other.
-
Solution structure of the Mesorhizobium loti K1 channel Cyclic Nucleotide-Binding Domain in complex with cAMP
EMBO reports, 2009Co-Authors: S Schunke, Matthias Stoldt, Kerstin Novak, U B Kaupp, Dieter WillboldAbstract:Cyclic nucleotide-sensitive ion channels, known as HCN and CNG channels, are crucial in neuronal excitability and signal transduction of sensory cells. HCN and CNG channels are activated by binding of Cyclic nucleotides to their intracellular Cyclic Nucleotide-Binding Domain (CNBD). However, the mechanism by which the binding of Cyclic nucleotides opens these channels is not well understood. Here, we report the solution structure of the isolated CNBD of a Cyclic nucleotide-sensitive K(+) channel from Mesorhizobium loti. The protein consists of a wide anti-parallel beta-roll topped by a helical bundle comprising five alpha-helices and a short 3(10)-helix. In contrast to the dimeric arrangement ('dimer-of-dimers') in the crystal structure, the solution structure clearly shows a monomeric fold. The monomeric structure of the CNBD supports the hypothesis that the CNBDs transmit the binding signal to the channel pore independently of each other.
S Schunke - One of the best experts on this subject based on the ideXlab platform.
-
resonance assignment of the ligand free Cyclic nucleotide binding Domain from the murine ion channel hcn2
Biomolecular Nmr Assignments, 2015Co-Authors: S Schunke, Matthias Stoldt, U B Kaupp, Justin Lecher, Claudia Borger, Friederike Winkhaus, Dieter WillboldAbstract:Hyperpolarization activated and Cyclic nucleotide-gated (HCN) ion channels as well as Cyclic nucleotide-gated (CNG) ion channels are essential for the regulation of cardiac cells, neuronal excitability, and signaling in sensory cells. Both classes are composed of four subunits. Each subunit comprises a transmembrane region, intracellular N- and C-termini, and a C-terminal Cyclic Nucleotide-Binding Domain (CNBD). Binding of Cyclic nucleotides to the CNBD promotes opening of both CNG and HCN channels. In case of CNG channels, binding of Cyclic nucleotides to the CNBD is sufficient to open the channel. In contrast, HCN channels open upon membrane hyperpolarization and their activity is modulated by binding of Cyclic nucleotides shifting the activation potential to more positive values. Although several high-resolution structures of CNBDs from HCN and CNG channels are available, the gating mechanism for murine HCN2 channel, which leads to the opening of the channel pore, is still poorly understood. As part of a structural investigation, here, we report the complete backbone and side chain resonance assignments of the murine HCN2 CNBD with part of the C-linker.
-
structural insights into conformational changes of a Cyclic nucleotide binding Domain in solution from mesorhizobium loti k1 channel
Proceedings of the National Academy of Sciences of the United States of America, 2011Co-Authors: S Schunke, Matthias Stoldt, U B Kaupp, Justin Lecher, Dieter WillboldAbstract:Cyclic nucleotide-sensitive ion channels, known as HCN and CNG channels, are activated by binding of ligands to a Domain (CNBD) located on the cytoplasmic side of the channel. The underlying mechanisms are not well understood. To elucidate the gating mechanism, structures of both the ligand-free and -bound CNBD are required. Several crystal structures of the CNBD from HCN2 and a bacterial CNG channel (MloK1) have been solved. However, for HCN2, the cAMP-free and -bound state did not reveal substantial structural rearrangements. For MloK1, structural information for the cAMP-free state has only been gained from mutant CNBDs. Moreover, in the crystal, the CNBD molecules form an interface between dimers, proposed to be important for allosteric channel gating. Here, we have determined the solution structure by NMR spectroscopy of the cAMP-free wild-type CNBD of MloK1. A comparison of the solution structure of cAMP-free and -bound states reveals large conformational rearrangement on ligand binding. The two structures provide insights on a unique set of conformational events that accompany gating within the ligand-binding site.
-
Resonance assignments of the nucleotide-free wildtype MloK1 Cyclic Nucleotide-Binding Domain
Biomolecular Nmr Assignments, 2010Co-Authors: S Schunke, U. Benjamin Kaupp, Matthias Stoldt, Justin Lecher, Dieter WillboldAbstract:Cyclic nucleotide-sensitive ion channels, known as HCN and CNG channels play crucial roles in neuronal excitability and signal transduction of sensory cells. These channels are activated by binding of Cyclic nucleotides to their intracellular Cyclic Nucleotide-Binding Domain (CNBD). A comparison of the structures of wildtype ligand-free and ligand-bound CNBD is essential to elucidate the mechanism underlying nucleotide-dependent activation of CNBDs. We recently reported the solution structure of the Mesorhizobium loti K1 (MloK1) channel CNBD in complex with cAMP. We have now extended these studies and achieved nearly complete assignments of 1H, 13C and 15N resonances of the nucleotide-free CNBD. A completely new assignment of the nucleotide-free wildtype CNBD was necessary due to the sizable chemical shift differences as compared to the cAMP bound CNBD and the slow exchange behaviour between both forms. Scattering of these chemical shift differences over the complete CNBD suggests that nucleotide binding induces significant overall conformational changes.
-
solution structure of the mesorhizobium loti k1 channel Cyclic nucleotide binding Domain in complex with camp
EMBO Reports, 2009Co-Authors: S Schunke, Matthias Stoldt, Kerstin Novak, U B Kaupp, Dieter WillboldAbstract:Cyclic nucleotide-sensitive ion channels, known as HCN and CNG channels, are crucial in neuronal excitability and signal transduction of sensory cells. HCN and CNG channels are activated by binding of Cyclic nucleotides to their intracellular Cyclic Nucleotide-Binding Domain (CNBD). However, the mechanism by which the binding of Cyclic nucleotides opens these channels is not well understood. Here, we report the solution structure of the isolated CNBD of a Cyclic nucleotide-sensitive K+ channel from Mesorhizobium loti. The protein consists of a wide anti-parallel β-roll topped by a helical bundle comprising five α-helices and a short 310-helix. In contrast to the dimeric arrangement (‘dimer-of-dimers') in the crystal structure, the solution structure clearly shows a monomeric fold. The monomeric structure of the CNBD supports the hypothesis that the CNBDs transmit the binding signal to the channel pore independently of each other.
-
Solution structure of the Mesorhizobium loti K1 channel Cyclic Nucleotide-Binding Domain in complex with cAMP
EMBO reports, 2009Co-Authors: S Schunke, Matthias Stoldt, Kerstin Novak, U B Kaupp, Dieter WillboldAbstract:Cyclic nucleotide-sensitive ion channels, known as HCN and CNG channels, are crucial in neuronal excitability and signal transduction of sensory cells. HCN and CNG channels are activated by binding of Cyclic nucleotides to their intracellular Cyclic Nucleotide-Binding Domain (CNBD). However, the mechanism by which the binding of Cyclic nucleotides opens these channels is not well understood. Here, we report the solution structure of the isolated CNBD of a Cyclic nucleotide-sensitive K(+) channel from Mesorhizobium loti. The protein consists of a wide anti-parallel beta-roll topped by a helical bundle comprising five alpha-helices and a short 3(10)-helix. In contrast to the dimeric arrangement ('dimer-of-dimers') in the crystal structure, the solution structure clearly shows a monomeric fold. The monomeric structure of the CNBD supports the hypothesis that the CNBDs transmit the binding signal to the channel pore independently of each other.
Giuseppe Melacini - One of the best experts on this subject based on the ideXlab platform.
-
Crystal structure of cGMP-dependent protein kinase Iβ Cyclic Nucleotide-Binding-B Domain : Rp-cGMPS complex reveals an apo-like, inactive conformation
FEBS letters, 2016Co-Authors: James C. Campbell, Bryan Vanschouwen, Giuseppe Melacini, Robin Lorenz, Friedrich W Herberg, Banumathi Sankaran, Choel KimAbstract:The R-diastereomer of phosphorothioate analogs of cGMP, Rp-cGMPS, is one of few known inhibitors of cGMP-dependent protein kinase I (PKG I); however, its mechanism of inhibition is currently not fully understood. Here, we determined the crystal structure of the PKG Iβ Cyclic Nucleotide-Binding Domain (PKG Iβ CNB-B), considered a 'gatekeeper' for cGMP activation, bound to Rp-cGMPS at 1.3 A. Our structural and NMR data show that PKG Iβ CNB-B bound to Rp-cGMPS displays an apo-like structure with its helical Domain in an open conformation. Comparison with the cAMP-dependent protein kinase regulatory subunit (PKA RIα) showed that this conformation resembles the catalytic subunit-bound inhibited state of PKA RIα more closely than the apo or Rp-cAMPS-bound conformations. These results suggest that Rp-cGMPS inhibits PKG I by stabilizing the inactive conformation of CNB-B.
-
Mechanism of cAMP Partial Agonism in Protein Kinase G (PKG)
The Journal of biological chemistry, 2015Co-Authors: Bryan Vanschouwen, Rajeevan Selvaratnam, Friedrich W Herberg, Rajanish Giri, Robin G. Lorenz, Choel Kim, Giuseppe MelaciniAbstract:Protein kinase G (PKG) is a major receptor of cGMP and controls signaling pathways often distinct from those regulated by cAMP. Hence, the selective activation of PKG by cGMP versus cAMP is critical. However, the mechanism of cGMP-versus-cAMP selectivity is only limitedly understood. Although the C-terminal Cyclic Nucleotide-Binding Domain B of PKG binds cGMP with higher affinity than cAMP, the intracellular concentrations of cAMP are typically higher than those of cGMP, suggesting that the cGMP-versus-cAMP selectivity of PKG is not controlled uniquely through affinities. Here, we show that cAMP is a partial agonist for PKG, and we elucidate the mechanism for cAMP partial agonism through the comparative NMR analysis of the apo, cGMP-, and cAMP-bound forms of the PKG Cyclic Nucleotide-Binding Domain B. We show that although cGMP activation is adequately explained by a two-state conformational selection model, the partial agonism of cAMP arises from the sampling of a third, partially autoinhibited state.
-
cAMP-dependent allostery and dynamics in Epac: An NMR view
Biochemical Society Transactions, 2012Co-Authors: Rajeevan Selvaratnam, Madoka Akimoto, Bryan Vanschouwen, Giuseppe MelaciniAbstract:Epac (exchange protein directly activated by cAMP) is a critical cAMP receptor, which senses cAMP and couples the cAMP signal to the catalysis of guanine exchange in the Rap substrate. In the present paper, we review the NMR studies that we have undertaken on the CBD (Cyclic-Nucleotide-Binding Domain) of Epac1. Our NMR investigations have shown that cAMP controls distal autoinhibitory interactions through long-range modulations in dynamics. Such dynamically mediated allosteric effects contribute not only to the cAMP-dependent activation of Epac, but also to the selectivity of Epac for cAMP in contrast with cGMP. In addition, we have mapped the interaction networks that couple the cAMP-binding site to the sites involved in the autoinhibitory interactions, using a method based on the covariance analysis of NMR chemical shifts. We anticipate that this approach is generally applicable to dissect allosteric networks in signalling Domains.
