Mode of action of cGMP-dependent protein kinase-specific inhibitors probed by photoaffinity cross-linking mass spectrometry.
The inhibitor peptide DT-2 (YGRKKRRQRRRPPLRKKKKKH) is the most potent and selective inhibitor of the cGMP-dependent protein kinase (PKG) known today. DT-2 is a construct of a PKG tight binding sequence (W45, LRKKKKKH, KI=0.8 microM) and a membrane translocating sequence (DT-6, YGRKKRRQRRRPP, KI=1.1 microM), that combined strongly inhibits PKG catalyzed phosphorylation (KI=12.5 nM) with approximately 1000-fold selectivity toward PKG over protein kinase A, the closest relative of PKG. However, the molecular mechanism behind this inhibition is not entirely understood. Using a combination of photoaffinity labeling, stable isotope labeling, and mass spectrometry, we have located the binding sites of PKG-specific substrate and inhibitor peptides. Covalent linkage of a PKG-specific substrate analogue was localized in the catalytic core on residues 356-372, also known as the glycine-rich loop, essential for ATP binding. By analogy, the individual inhibitor peptides W45 and DT-6 were also found to cross-link near the glycine-rich loop, suggesting these are both substrate competitive inhibitors. A bifunctional photoreactive analogue of DT-2 was found to generate dimers of PKG. This cross-linking induced covalent PKG dimerization was not observed for an N-terminal deletion mutant of PKG, which lacks the dimerization domain. In addition, non-covalent mass spectrometry was used to determine binding stoichiometry and binding order of the inhibitor peptides. Dimeric PKG binds two W45 and DT-6 peptides, whereas only one DT-2 molecule was observed to bind to the dimeric PKG. Taken together, these findings imply that (i) the two individual components making up DT-2 are both targeted against the substrate-binding site and (ii) binding of a single DT-2 molecule inactivates both PKG monomers simultaneously, which is an indication that (iii) in cGMP-activated PKG the catalytic centers of both subunits may be in each other's proximity.
From the Biomolecular Mass Spectrometry and Proteomics Group, Bijvoet Center for Biomolecular Research and Utrecht Institute for Pharmaceutical Sciences, Utrecht University, Sorbonnnelaan 16, Utrecht 3584 CA, The Netherlands; Department of Biotechnology, Delft, University of Technology, Delft 2628 BC, The Netherlands.
Martijn W H
MWH
Pinkse
Medicinal Chemistry and Chemical Biology, Utrecht Institute for Pharmaceutical Sciences, Department of Pharmaceutical Sciences, Faculty of Science, Utrecht University, Utrecht 3584 CA, The Netherlands.
Dirk T S
DTS
Rijkers
Department of Pharmacology, College of Medicine, University of Vermont, Burlington, Vermont 05405.
Wolfgang R
WR
Dostmann
From the Biomolecular Mass Spectrometry and Proteomics Group, Bijvoet Center for Biomolecular Research and Utrecht Institute for Pharmaceutical Sciences, Utrecht University, Sorbonnnelaan 16, Utrecht 3584 CA, The Netherlands. Electronic address: a.j.r.heck@uu.nl.
Albert J R
AJR
Heck
D000818:Animals; D001665:Binding Sites; D002478:Cells, Cultured; D003432:Cross-Linking Reagents / Q000494:pharmacology; D017868:Cyclic AMP-Dependent Protein Kinases / Q000737:chemistry; D017869:Cyclic GMP-Dependent Protein Kinases / Q000037:antagonists & inhibitors* / Q000737:chemistry; D019281:Dimerization; D005452:Fluoresceins / Q000494:pharmacology*; D013058:Mass Spectrometry / Q000379:methods*; D010446:Peptide Fragments / Q000494:pharmacology*; D019920:Photoaffinity Labels / Q000494:pharmacology*; D047428:Protein Kinase Inhibitors / Q000494:pharmacology; D017434:Protein Structure, Tertiary; D018411:Spodoptera
D003432:Cross-Linking Reagents; C513505:DT-2 compound; D005452:Fluoresceins; D010446:Peptide Fragments; D019920:Photoaffinity Labels; D047428:Protein Kinase Inhibitors; C518603:protein kinase inhibitor peptide (5-24); D017868:Cyclic AMP-Dependent Protein Kinases; D017869:Cyclic GMP-Dependent Protein Kinases