Abstract
Thermodynamic parameters of interactions of calcium-saturated calmodulin (Ca(2+)-CaM) with melittin, C-terminal fragment of melittin, or peptides derived from the CaM binding regions of constitutive (cerebellar) nitric-oxide synthase, cyclic nucleotide phosphodiesterase, calmodulin-dependent protein kinase I, and caldesmon (CaD-A, CaD-A*) have been measured using isothermal titration calorimetry. The peptides could be separated into two groups according to the change in heat capacity upon complex formation, DeltaC(p). The calmodulin-dependent protein kinase I, constitutive (cerebellar) nitric-oxide synthase, and melittin peptides have DeltaC(p) values clustered around -3.2 kJ.mol(-1).K(-1), consistent with the formation of a globular CaM-peptide complex in the canonical fashion. In contrast, phosphodiesterase, the C-terminal fragment of melittin, CaD-A, and CaD-A* have DeltaC(p) values clustered around -1.6 kJ.mol(-1).K(-1), indicative of interactions between the peptide and mostly one lobe of CaM, probably the C-terminal lobe. It is also shown that the interactions for different peptides with Ca(2+)-CaM can be either enthalpically or entropically driven. The difference in the energetics of peptide/Ca(2+)-CaM complex formation appears to be due to the coupling of peptide/Ca(2+)-CaM complex formation to the coil-helix transition of the peptide. The binding of a helical peptide to Ca(2+)-CaM is dominated by favorable entropic effects, which are probably mostly due to hydrophobic interactions between nonpolar groups of the peptide and Ca(2+)-CaM. Applications of these findings to the design of potential CaM inhibitors are discussed.
Highlights
Understanding detailed molecular mechanisms that govern macromolecular interactions represents one of the major goals of structural biology
It could be that the increase in solvent entropy is offset by other phenomena, such as the loss of mobility of some amino acid side chains [20, 21] or the loss of with an extra C-terminal cysteine residue; CaD-B1, peptide of residues 674 – 696 of CaD; calmodulin-dependent protein kinase I (CaMKI), CaM-dependent protein kinase I; cerebellar nitric-oxide synthase (cNOS), constitutive nitric-oxide synthase; ITC, isothermal titration calorimetry; MEL, melittin; MLC, C-terminal 13-residue fragment of MEL; MLCK, skeletal muscle myosin light chain kinase; MOPS, 3-(Nmorpholino)propanesulfonic acid; PDE, 3Ј:5Ј-cyclic nucleotide phosphodiesterase; PIPES, 1,1-piperazinediethanesulfonic acid; smMLCK, smooth muscle myosin light chain kinase; ASA, accessible surface area; LPE, linked protonation effects
Where ⌬Hb is the enthalpy of binding per se and ⌬nϩ1⁄7⌬Hion is the enthalpy of linked protonation effects (LPE)
Summary
Understanding detailed molecular mechanisms that govern macromolecular interactions represents one of the major goals of structural biology. The alignment of the helices within each lobe changes upon Ca2ϩ binding, resulting in the exposure of two methionine-rich hydrophobic “patches,” one in each lobe [5, 6] These hydrophobic patches enable CaM in the Ca2ϩ-loaded form (Ca2ϩ-CaM) to interact with a number of intracellular proteins and enzymes that are involved in a wide variety of different biochemical processes [7,8,9]. In this paper we report the results of the direct calorimetric measurements of thermodynamics of complex formation of nine different peptides with Ca2ϩ-CaM These peptides were derived from the CaM-binding sequences of CaM-dependent protein kinase I [23], cyclic nucleotide phosphodiesterase [22], caldesmon (24 –26), constitutive cerebellar nitric-oxide synthase [27, 28], and bee venom melittin.. Analysis of the thermodynamic data for CaM-peptide complex formation allowed us to shed more light on the nature of physical forces underlying Ca2ϩ-CaM/ target interactions
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