Interdomain cooperativity of calmodulin bound to melittin preferentially increases calcium affinity of sites I and II.
Calmodulin (CaM) is the primary transducer of calcium fluxes in eukaryotic cells. Its two domains allosterically regulate myriad target proteins through calcium-linked association and conformational change. Many of these proteins have a basic amphipathic alpha-helix (BAA) motif that binds one or both CaM domains. Previously, we demonstrated domain-specific binding of melittin, a model BAA peptide, to Paramecium CaM (PCaM): C-domain mutations altered the interaction with melittin, whereas N-domain mutations had no discernable effect. Here, we report on the use of fluorescence and NMR spectroscopy to measure the domain-specific association of melittin with calcium-saturated ((Ca(2+))(4)-PCaM) or calcium-depleted (apo) PCaM, which has enabled us to determine the free energies of calcium binding to the PCaM-melittin complex, and to estimate interdomain cooperativity. Under apo conditions, melittin associated with each PCaM domain fragment (PCaM(1-80) and PCaM(76-148)), as well as with the C-domain of full-length PCaM (PCaM(1-148)). In the presence of calcium, all of these interactions were again observed, in addition to which an association with the N-domain of (Ca(2+))(4)-PCaM(1-148) occurred. This new association was made possible by the fact that melittin changed the calcium-binding preferences for the domains from sequential (C > N) to concomitant, decreasing the median ligand activity of calcium toward the N-domain 10-fold more than that observed for the C-domain. This selectivity may be explained by a free energy of cooperativity of -3 kcal/mol between the N- and C-domains. This study demonstrates multiple domain-selective differences in the interactions between melittin and PCaM. Our findings support a model that may apply more generally to ion channels that associate with the C-domain of CaM under low (resting) calcium conditions, but rearrange when calcium binding triggers an association of the N- domain with the channel.
Newman
Rhonda A
RA
Department of Biochemistry, Roy J. and Lucille A. Carver College of Medicine, University of Iowa, Iowa City, Iowa 52242-1109, USA.
Van Scyoc
Wendy S
WS
Sorensen
Brenda R
BR
Jaren
Olav R
OR
Shea
Madeline A
MA
D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D002118:Calcium / Q000378:metabolism*; D002147:Calmodulin / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D017124:Conserved Sequence; D005454:Fluorescence Polarization; D057927:Hydrophobic and Hydrophilic Interactions; D007700:Kinetics; D008555:Melitten / Q000737:chemistry / Q000378:metabolism*; D008954:Models, Biological; D008958:Models, Molecular; D008969:Molecular Sequence Data; D019906:Nuclear Magnetic Resonance, Biomolecular; D010247:Paramecium / Q000235:genetics / Q000378:metabolism; D011485:Protein Binding; D011487:Protein Conformation; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D013050:Spectrometry, Fluorescence; D013816:Thermodynamics; D014364:Tryptophan / Q000737:chemistry
D002147:Calmodulin; D011994:Recombinant Proteins; D008555:Melitten; D014364:Tryptophan; D002118:Calcium