Structural changes in melittin and calmodulin upon complex formation and their modulation by calcium

Abstract

In the presence of Ca2+, calmodulin formed a 1:1 high-affinity complex (Kd = 3 nM) with melittin, a peptide from bee venom; in the presence of EDTA, a 2nd type of complex, of much lower affinity, was formed. These interactions were studied by tryptophan fluorescence and circular dichroism spectroscopy in near- and far-UV. Interaction between the 2 peptides in the presence or absence of Ca2+ led to the sheilding of the tryptophan residue of melittin from its aqueous environment and to an increase in the .alpha.-helical content of bound melittin. The Ca2+-dependent high-affinity complex formation enhanced the .alpha.-helical content of melittin from 5-72%. Provided Ca2+ was present, the interaction between the 2 peptides led to significant changes in the environment of at least 1 tyrosine residue of calmodulin as measured by near-UV circular dichroism. In the absence of Ca2+, calmodulin bound 2 melittin molecules with a Kd of .apprx. 10 .mu.M; at higher concentrations of free melittin, additional binding occurred (up to 5 mol melittin/mol calmodulin), with concomitant denaturation of calmodulin. In the presence of 4.0 M urea, the low-affinity complexes formed in the absence of Ca2+ dissociated due to the denaturation of metal-free calmodulin; the spectroscopic signals of the high-affinity Ca2+-dependent complex were not affected. Equilibrium dialysis on a equimolar mixture of melittin and calmodulin showed that melittin enhanced the affinity of calmodulin for Ca2+, resulting in the following macroscopic Kd: K1 = 0.2 .mu.M; K2 = 0.72 .mu.M; K3 = 0.18 .mu.M; K4 = 1.1 .mu.M. The energy coupling involved in the formation of the ternary complex amounted to at least 7.9 kcal/mol, indicating that the saturation of calmodulin by Ca2+ increased its affinity for melittin at least (1.5 .times. 106)-fold. In the presence of Ca2+, melittin bound to that part of the surface of calmodulin that also interacted with the target enzyme.