Mechanism of cAMP Partial Agonism in Protein Kinase G (PKG)

VanSchouwen, Bryan; Selvaratnam, Rajeevan; Giri, Rajanish; Lorenz, Robin; Herberg, Friedrich W.; Kim, Choel; Melacini, Giuseppe

doi:10.1074/jbc.m115.685305

Cited by 47 publications

(53 citation statements)

References 50 publications

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“…Specifically, NMR peak positions reflect populationweighted averages of the respective ppm values for the pure inactive and active CBD states, which exchange rapidly on the chemical shift NMR time scale (9,40). A simple but effective means to extract state populations from average chemical shifts is the projection analysis (9,21,28,29,30,41), in which changes in HSQC cross-peak positions arising from the mutation are evaluated relative to a reference vector defined by the WT apo and WT holo samples. For example, Fig.…”

Section: C E and F)mentioning

confidence: 99%

“…It is still not clear how the S672R mutation perturbs the apo-CBD, and in general the CBD dynamics, which has been recognized as a key determinant of the auto-inhibitory CBD function in HCN gating and in other cAMP-dependent systems (9,13,(21)(22)(23)(24)(25)(26)(27)(28)(29). One of the aspects of CBD dynamics that is most relevant for auto-inhibition is the equilibrium between inactive and active conformations (Fig.…”

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confidence: 99%

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Free energy landscape remodeling of the cardiac pacemaker channel explains the molecular basis of familial sinus bradycardia

Boulton¹,

Akimoto

Akbarizadeh

et al. 2017

Journal of Biological Chemistry

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Edited by Norma AllewellThe hyperpolarization-activated and cyclic nucleotide-modulated ion channel (HCN) drives the pacemaker activity in the heart, and its malfunction can result in heart disorders. One such disorder, familial sinus bradycardia, is caused by the S672R mutation in HCN, whose electrophysiological phenotypes include a negative shift in the channel activation voltage and an accelerated HCN deactivation. The outcomes of these changes are abnormally low resting heart rates. However, the molecular mechanism underlying these electrophysiological changes is currently not fully understood. Crystallographic investigations indicate that the S672R mutation causes limited changes in the structure of the HCN intracellular gating tetramer, but its effects on protein dynamics are unknown. Here, we utilize comparative S672R versus WT NMR analyses to show that the S672R mutation results in extensive perturbations of the dynamics in both apo-and holo-forms of the HCN4 isoform, reflecting how S672R remodels the free energy landscape for the modulation of HCN4 by cAMP, i.e. the primary cyclic nucleotide modulator of HCN channels. We show that the S672R mutation results in a constitutive shift of the dynamic auto-inhibitory equilibrium toward inactive states of HCN4 and broadens the free-energy well of the apo-form, enhancing the millisecond to microsecond dynamics of the holo-form at sites critical for gating cAMP binding. These S672R-induced variations in dynamics provide a molecular basis for the electrophysiological phenotypes of this mutation and demonstrate that the pathogenic effects of the S672R mutation can be rationalized primarily in terms of modulations of protein dynamics.

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Section: C E and F)mentioning

confidence: 99%

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confidence: 99%

Free energy landscape remodeling of the cardiac pacemaker channel explains the molecular basis of familial sinus bradycardia

Boulton¹,

Akimoto

Akbarizadeh

et al. 2017

Journal of Biological Chemistry

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“…). Although complete assignment of the PBC in all three forms (i.e., apo, cGMP‐, and Rp‐cGMPS‐bound) was not possible for all PBC amino acids due to line‐broadening and/or peak overlap, the assigned NMR cross‐peaks indicate that in solution, the Rp‐bound PBC exists in a distinct conformation from that of the cGMP‐bound state . For example, Ala309 showed a proton shift from ~ 8.5 p.p.m.…”

Section: Resultsmentioning

confidence: 99%

Crystal structure of cGMP‐dependent protein kinase Iβ cyclic nucleotide‐binding‐B domain : Rp‐cGMPS complex reveals an apo‐like, inactive conformation

et al. 2016

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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 Å. 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.

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“…For example, amide 15 N and 1 H chemical shifts are hypersensitive to conformational variations, which poses a major challenge for their computational prediction and for their use in chemical shift-based structure determination but makes them an ideal atomic-resolution probe to sense the effect of allosteric perturbations through comparative NMR analyses (3, 6). The latter include not only conventional apo vs. holo or WT vs. mutant binary chemical shift comparisons, but they extend also to ternary or multiperturbation vectorial, correlative, and statistical analyses (6)(7)(8)(9)(10)(11)(12)(13).…”

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confidence: 99%

“…1) (6-16). However, when allostery involves exchange between more than two states, deviations from these linear patterns are observed at least for selected residues (8). Hence, pattern analysis of chemical shift variations provides an effective means to identify subsectors of residues that exhibit concerted responses to allosteric perturbations (7).…”

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confidence: 99%