High-affinity formation of a 2:1 complex between gramicidin S and calmodulin

Abstract

Two molecules of gramicidin S, a very rigid cyclic decapeptide rich in beta-sheet structure, can bind in a Ca2+-dependent way to a calmodulin molecule in the presence as well as in the absence of 4 M-urea. The flow-microcalorimetric titration of 25 microM-calmodulin with gramicidin S at 25 degrees C is endothermic for 21.3 kJ.mol-1; the enthalpy change is strictly linear up to a ratio of 2, indicating that the affinity constant for binding of the second gramicidin S is at least 10(7) M-1. In 4 M-urea the peptide quantitatively displaces seminalplasmin from calmodulin, as monitored by tryptophan fluorescence. An iterative data treatment of these competition experiments revealed strong positive co-operativity with K1 less than 5 X 10(5) M-1 and K1.K2 = 2.8 X 10(12) M-2. A competition assay with the use of immobilized melittin enabled us to monitor separately the binding of the second gramicidin S molecule: the K2 value is 1.9 X 10(7) M-1. By complementarity, the K1 value is 1.5 X 10(5) M-1. In the absence of urea the seminalplasmin displacement is incomplete: the data analysis shows optimal fitting with K1 less than 2 X 10(4) M-1 and K1.K2 = 3.2 X 10(11) M-2 and reveals that the mixed complex (calmodulin-seminalplasmin-gramicidin S) is quite stable and is even not fully displaced from calmodulin at high concentrations of gramicidin S. The activation of bovine brain phosphodiesterase by calmodulin is not impaired up to 0.2 microM-gramicidin S. According to our model the ternary complex enzyme-calmodulin-gramicidin is relatively important and displays the same activity as the binary complex enzyme-calmodulin. Gramicidin S also displaces melittin from calmodulin synergistically, as monitored by c.d. Our studies with gramicidin S reveal the importance of multipoint attachments in interactions involving calmodulin and confirm the heterotropic co-operativity in the binding of calmodulin antagonists first demonstrated by Johnson [(1983) Biochem. Biophys. Res. Commun. 112, 787-793].