Thermodynamics of calmodulin trapping by Ca2+/calmodulin-dependent protein kinase II: subpicomolar Kd determined using competition titration calorimetry.
Calmodulin (CaM) trapping by Ca2+/calmodulin-dependent protein kinase II (CaMKII) is a phenomenon whereby the affinity of CaM for CaMKII increases >1000-fold following CaMKII autophosphorylation. The molecular basis of this effect is not entirely understood. Binding of CaM to the phosphorylated and the unphosphorylated states of CaMKII is well mimicked by the interaction of CaM with two different length peptides taken from the CaM-binding region of CaMKII, peptides we refer to as the long and intermediate peptides. To better understand the conformational change accompanying CaM trapping, we have used isothermal titration calorimetry (ITC) to compare the binding thermodynamics of CaM to these peptides as well as to a shorter CaMKII-based peptide. Calorimetric analysis revealed that the enthalpy, rather than the entropy, distinguished binding of these three peptides. Furthermore, the heat capacity change was found to be similar for the long and intermediate peptides but smaller in magnitude for the short peptide. Direct titration of CaM with peptide provided the Kd value for the short peptide (Kd = 5.9 +/- 2.4 microM), but a novel, two-phased competitive binding strategy was necessary to ascertain the affinities of the intermediate (Kd = 0.17 +/- 0.06 nM) and long (Kd = 0.07 +/- 0.04 pM) peptides. To our knowledge, the Kd for the long peptide is the most potent measured to date using ITC. Together, the findings reported here support a model whereby the final conformational change accompanying CaM trapping buries little additional surface area but does involve formation of new hydrogen bonds and van der Waals contacts that contribute to formation of the high-affinity, CaM-trapped state.
Tse
Joyce K Y
JK
Department of Biochemical Pharmacology, Roche Palo Alto LLC, 3431 Hillview Avenue, Palo Alto, California 94304, USA.
Giannetti
Anthony M
AM
Bradshaw
J Michael
JM
D000818:Animals; D001665:Binding Sites; D001667:Binding, Competitive; D054732:Calcium-Calmodulin-Dependent Protein Kinase Type 2; D017871:Calcium-Calmodulin-Dependent Protein Kinases / Q000378:metabolism*; D002147:Calmodulin / Q000378:metabolism*; D002151:Calorimetry; D007700:Kinetics; D008958:Models, Molecular; D010446:Peptide Fragments / Q000378:metabolism; D010766:Phosphorylation; D011487:Protein Conformation; D051381:Rats; D013816:Thermodynamics
D002147:Calmodulin; D010446:Peptide Fragments; D054732:Calcium-Calmodulin-Dependent Protein Kinase Type 2; D017871:Calcium-Calmodulin-Dependent Protein Kinases