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Interdomain cooperativity of calmodulin bound to melittin preferentially increases calcium affinity of sites I and II
Author(s) -
Newman Rhonda A.,
Van Scyoc Wendy S.,
Sorensen Brenda R.,
Jaren Olav R.,
Shea Madeline A.
Publication year - 2008
Publication title -
proteins: structure, function, and bioinformatics
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.699
H-Index - 191
eISSN - 1097-0134
pISSN - 0887-3585
DOI - 10.1002/prot.21861
Subject(s) - melittin , calmodulin , cooperativity , chemistry , calcium , biophysics , calcium binding protein , biochemistry , crystallography , peptide , biology , organic chemistry
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 α‐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. Proteins 2008. © 2008 Wiley‐Liss, Inc.