Probing noncovalent protein-ligand interactions of the cGMP-dependent protein kinase using electrospray ionization time of flight mass spectrometry.
Nanoflow electrospray ionization time of flight mass spectrometry (ESI-TOF-MS) was used to study activation properties of the cGMP-dependent protein kinase (PKG). Our nanoflow ESI-TOF-MS analysis confirms that PKG mainly occurs as a 153 kDa homodimer and is able to bind four cGMP molecules, which is in agreement with the known stoichiometry. Binding order and stoichiometry of cGMP, the non-hydrolysable ATP analog beta,gamma-imidoadenosine 5'-triphosphate (AMPPNP) and Mn2+ for PKG were characterized as model for the active PKG-cGMP-ATP/Mg2+ complex. Already in the absence of cGMP, a noncovalent complex between PKG and two molecules of AMPPNP could be observed by ESI-TOF-MS. Binding of AMPPNP to PKG was strongly enhanced by the addition of MnCl2 to the spray solution. This is in agreement with binding of AMPPNP/Mn2+ in the ATP binding pocket of PKG since all protein kinases require a metal ion to accompany ATP in the ATP-binding pocket for proper positioning of the beta and gamma phosphates. Additionally, this finding could imply that within the inactive conformation of PKG, the autoinhibition-domain, when in contact with the substrate-docking domain, does not block the entrance to the ATP-binding site. In the presence of cGMP, less of the fully saturated PKG-(cGMP)4(AMPPNP/Mn2+)2 complex was observed, suggesting that the PKG-ATP interaction is weakened in the active conformation of PKG. Additionally, limited proteolysis in combination with native-ESI MS showed to be a useful tool to study the contact regions on the PKG-dimer and also allowed the rapid determination of the overall autophosphorylation status of the protein. These measurements indicated that autophosphorylation mainly occurs within the first 80 aminoterminal residues and involves in total 3-4 phosphates per subunit.
Department of Biomolecular Mass Spectrometry, Bijvoet Center for Biomolecular Research and Utrecht Institute for Pharmaceutical Sciences, Utrecht University, Sorbonnelaan 16, 3584 CA, Utrecht, The Netherlands. m.w.h.pinkse@pharm.uu.nl.
Martijn W H
MWH
Pinkse
Department of Biomolecular Mass Spectrometry, Bijvoet Center for Biomolecular Research and Utrecht Institute for Pharmaceutical Sciences, Utrecht University, Sorbonnelaan 16, 3584 CA, Utrecht, The Netherlands.
Albert J R
AJR
Heck
Pfizer Global Research and Development, Sandwich, United Kingdom.
Klaus
K
Rumpel
Pfizer Global Research and Development, Sandwich, United Kingdom.
Frank
F
Pullen
D000266:Adenylyl Imidodiphosphate / Q000378:metabolism; D000818:Animals; D002417:Cattle; D006152:Cyclic GMP / Q000378:metabolism; D017869:Cyclic GMP-Dependent Protein Kinases / Q000737:chemistry* / Q000378:metabolism*; D008024:Ligands; D008345:Manganese / Q000378:metabolism; D008958:Models, Molecular; D011485:Protein Binding; D011487:Protein Conformation; D021241:Spectrometry, Mass, Electrospray Ionization
D008024:Ligands; D000266:Adenylyl Imidodiphosphate; D008345:Manganese; D017869:Cyclic GMP-Dependent Protein Kinases; D006152:Cyclic GMP