The hinge region operates as a stability switch in cGMP-dependent protein kinase I alpha.
The molecular mechanism of cGMP-dependent protein kinase activation by its allosteric regulator cyclic-3',5'-guanosine monophosphate (cGMP) has been intensely studied. However, the structural as well as thermodynamic changes upon binding of cGMP to type I cGMP-dependent protein kinase are not fully understood. Here we report a cGMP-induced shift of Gibbs free enthalpy (DeltaDeltaGD) of 2.5 kJ.mol-1 as determined from changes in tryptophan fluorescence using urea-induced unfolding for bovine PKG Ialpha. However, this apparent increase in overall stability specifically excluded the N-terminal region of the kinase. Analyses of tryptic cleavage patterns using liquid chromatography-coupled ESI-TOF mass spectrometry and SDS/PAGE revealed that cGMP binding destabilizes the N-terminus at the hinge region, centered around residue 77, while the C-terminus was protected from degradation. Furthermore, two recombinantly expressed mutants: the deletion fragment Delta1-77 and the trypsin resistant mutant Arg77Leu (R77L) revealed that the labile nature of the N-terminus is primarily associated with the hinge region. The R77L mutation not only stabilized the N-terminus but extended a stabilizing effect on the remaining domains of the enzyme as well. These findings support the concept that the hinge region of PKG acts as a stability switch.
Scholten
Arjen
A
Department of Pharmacology, College of Medicine, University of Vermont, Burlington, VT 05405, USA.
Fuss
Hendrik
H
Heck
Albert J R
AJ
Dostmann
Wolfgang R
WR
D000595:Amino Acid Sequence; D019943:Amino Acid Substitution / Q000235:genetics; D000818:Animals; D002417:Cattle; D002460:Cell Line; D006152:Cyclic GMP / Q000737:chemistry* / Q000378:metabolism; D062965:Cyclic GMP-Dependent Protein Kinase Type I; D017869:Cyclic GMP-Dependent Protein Kinases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D004795:Enzyme Stability / Q000235:genetics; D010446:Peptide Fragments / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D011485:Protein Binding; D017434:Protein Structure, Tertiary / Q000235:genetics; D017384:Sequence Deletion; D013816:Thermodynamics; D014364:Tryptophan / Q000737:chemistry
D010446:Peptide Fragments; D014364:Tryptophan; D062965:Cyclic GMP-Dependent Protein Kinase Type I; D017869:Cyclic GMP-Dependent Protein Kinases; D006152:Cyclic GMP