Thermodynamic characterization of the interaction of human cyclophilin 18 with cyclosporin A.
Isothermal titration calorimetry (ITC) was used to investigate thermodynamic parameters of the cyclosporin A (CsA)-cyclophilin 18 (hCyp18) association reaction. We have calculated the thermodynamic parameters (enthalpy, entropy, heat capacity, and free energy of binding) of the CsA/hCyp18 complexation. All but two methods described in the literature underestimate the affinity to hCyp18 of CsA. We found that the association constant (1.1.10(8) M(-1) at 10 degrees C) of CsA to hCyp18 is in close agreement with the reciprocal of the reported inhibitory constant of the peptidylprolyl cis/trans isomerase activity of hCyp18. Interpretation of the thermodynamic parameters in buffered solution of water, 30% glycerol and D(2)O leads to the conclusion that the highly specific binding of CsA to hCyp18 is mainly mediated through hydrogen bonding and to a lesser degree through hydrophobic interaction. Furthermore, the pH dependence of the association constant was determined and analyzed according to a single proton linkage model, resulting in a pK(a) value of 5.7 in free hCyp18 and below 4.5 in the CsA complexed form. Titration experiments using different single component buffers possessing different heats of ionization allowed us to estimate that statistically half a proton is transferred upon CsA binding from the binding interface of hCyp18 to the buffer at pH 5.5. No proton transfer was detected at pH 7.5. The thermodynamic results are discussed in relation to the published X-ray and NMR structure of the free and CsA complexed hCyp18.
Fanghänel
Jörg
J
Max Planck Research Unit for Enzymology of Protein Folding, Weinbergweg 22, D-06120 Halle (Saale), Germany.
Fischer
Gunter
G
D000465:Algorithms; D002021:Buffers; D002151:Calorimetry; D002942:Circular Dichroism; D021983:Cyclophilins / Q000096:biosynthesis / Q000737:chemistry*; D016572:Cyclosporine / Q000737:chemistry*; D003903:Deuterium / Q000737:chemistry; D017666:Deuterium Oxide; D004926:Escherichia coli / Q000378:metabolism; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D011485:Protein Binding; D012997:Solvents; D013056:Spectrophotometry, Ultraviolet; D013816:Thermodynamics; D014783:Viscosity
D002021:Buffers; D012997:Solvents; D016572:Cyclosporine; D003903:Deuterium; D021983:Cyclophilins; D017666:Deuterium Oxide