Melittin binding causes a large calcium-dependent conformational change in calmodulin.

Abstract

The interaction between calmodulin and its target protein is a key step in many calcium-regulated cellular functions. Melittin binds tightly to calmoduiln in the presence of calcium and is a competitive inhibitor of codulin function. Using melittin as a model for the target peptide of calmodulin, we have found a large Ca2+-dependent conformational change of almoulin in solution induced by peptide binding. Mg+ does not substitute for Ca2+ in producing the conformation change. Small-angle x-ray scattering has shown that calmodulin exists as a dumbbell in solution, simllar to that observed in the crystalline state. Our present measurements reveal that the overall structure of the Ca2+-calmodulinmelittin complex is not a dumbbell but a globular shape. Upon binding melittin, the radius of gyration decreases from 20.9 to 18.0 A and the largest dimension decreases from 60 to 47.5 A.In the absence of calcium, however, melittin has little effect on the solution structure of calmodulin. Because of the importance of calmodulin-enzyme interactions, much work has focused on the molecular mechanisms underlying the protein-protein interaction and the ensuing enzyme activation. The crystal structure of calmodulin (2-4) has provided the structural framework for numerous studies.In crystals, calmodulin and its close homologue troponin C adopt an unusual dumbbell shape (2-6). The two lobes of the dumbbell are connected by a central helix. Because of its unusual shape and high surface/volume ratio, this structure has raised many questions related to the form and function of the protein, including the relationship between the crystal and solution structures and the possibility of alternative conformations. Some ofthese questions have been addressed through techniques such as small-angle x-ray scattering (7,8). From small-angle x-ray scattering data, information about molecular size and shape can be obtained from protein molecules in solution, thus providing complementary data to those obtained from crystallographic methods. While small-angle x-ray scattering is a much lower resolution technique than x-ray crystallography, a major advantage, in addition to the ability to measure proteins in solution, is that samples can be studied under near-physiological conditions, which is frequently impossible for protein crystals.Differences between solution and crystal structures of calmodulin seem most likely to involve dynamic aspects of the spatial relationship of the two lobes of the molecule, governed by flexibility of the central helical region. Previous small-angle x-ray scattering studies on calmodulin (7, 8) have indicated that in solution the protein possesses an elongated bibbed shape, generally consistent with the crystal structure. However, such measurements provide information only on the time-averaged structure and do not preclude the possibility that flexibility in the helix may exist. The consequences of such flexibility, particularly in terms of its role in the interactions of calmodulin with its target proteins, have ...

Hydroxyl radical probe of the calmodulin-melittin complex interface by electrospray ionization mass spectrometry

“…Fluorescence and circular dichroism spectroscopy have revealed that in the presence of calcium, both calmodulin and melittin undergo significant structural changes, particularly in the vicinity of tyrosine residues 99 and 138 in calmodulin and at tryptophan 19 in melittin [9]. Small angle X-ray scattering has also identified significant structural changes in calmodulin with its transformation from a dumbbell to globular conformation upon its binding to melittin [13]. Similar conclusions have been drawn from limited proteolysis experiments followed by mass spectrometric analysis [16].…”

CBP501-Calmodulin Binding Contributes to Sensitizing Tumor Cells to Cisplatin and Bleomycin

“…3F-H). When we examined melittin, a peptidic inhibitor of CaM from bee venom (18), the combined treatment with melittin/cisplatin showed relative increases in the G 2 -M population (Fig. 4I).…”

Structural and Functional Roles of Modules in Hemoglobin

“…The proteins are dissolved in the buffer solution with 10 mM Tris (pH 7.4), 10 mM NaCN. Each plot was obtained by the extrapolation to zero protein concentration of the data for four different protein concentrations (23). For clarity, each plot is shifted on the ln(I(Q)) axis.…”

“…The scattering curve at infinite dilution was obtained from a series of scattering data with different protein concentrations (23). X-ray scattering intensities at the small angle region are given as the equation,…”