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cGMP-binding prepares PKG for substrate binding by disclosing the C-terminal domain.

Vera Alverdi1, Hortense Mazon, Cees Versluis

  • 1Department of Biomolecular Mass Spectrometry, Bijvoet Center for Biomolecular Research and Utrecht Institute for Pharmaceutical Sciences, Utrecht University, Sorbonnelaan 16, 3584 CA Utrecht, The Netherlands.

Journal of Molecular Biology
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Summary

Cyclic guanosine monophosphate-dependent protein kinase (PKG) undergoes structural changes upon activation. Mass spectrometry revealed that cGMP binding exposes the substrate region, offering new insights into PKG

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Area of Science:

  • Biochemistry
  • Molecular Biology
  • Cell Signaling

Background:

  • Type I cyclic guanosine 3',5'-monophosphate (cGMP)-dependent protein kinase (PKG) is a key enzyme in the nitric oxide/cGMP pathway.
  • PKG acts as a major intracellular cGMP receptor, regulating cellular functions like smooth muscle relaxation and synaptic plasticity.
  • The three-dimensional structure of PKG remains elusive, hindering a full understanding of its cGMP-dependent activation mechanism.

Purpose of the Study:

  • To investigate the structural alterations in PKG I alpha upon cGMP activation.
  • To elucidate the conformational changes associated with PKG activation using a high-resolution technique.

Main Methods:

  • Mass spectrometry-based hydrogen/deuterium exchange (HDX-MS) experiments were employed.
  • Site-specific deuterium exchange measurements were performed on PKG I alpha.

Main Results:

  • cGMP binding induced significant structural changes in PKG I alpha.
  • The autoinhibitory domain and hinge region became more solvent-exposed.
  • cGMP-binding domains were more protected, and unexpectedly, the substrate-binding region of holo-PKG was also exposed.

Conclusions:

  • HDX-MS provides detailed insights into PKG structural dynamics.
  • The observed structural changes, particularly the exposure of the substrate-binding region, shed new light on the kinase activation process of PKG.
  • These findings contribute to understanding the molecular mechanisms underlying PKG-mediated cellular signaling.