The effect of calcium ions and peptide ligands on the relative stabilities of the calmodulin dumbbell and compact structures.
A combination of ion mobility and mass spectrometry methods was used to characterize the molecular shape of the protein calmodulin (CaM) and its complexes with calcium and a number of peptide ligands. CaM, a calcium-binding protein composed of 148 amino acid residues, was found by X-ray crystallography to occur both in a globular shape and in the shape of an extended dumbbell. Here, it was found, as solutions of CaM and CaM complexes were sprayed into the solvent-free environment of the mass spectrometer, that major structural features of the molecule and the stoichiometry of the units constituting a complex in solution were preserved in the desolvation process. Two types of CaM structures were observed in our experiments: a compact and an extended form of CaM with measured cross sections in near-perfect agreement with those calculated for the known globular and extended dumbbell X-ray geometries. Calcium-free solutions yielded predominantly an extended CaM conformation. Ca(n)(2+)-CaM complexes were observed in calcium-containing solutions, n = 0-4, with the population of the compact conformation increasing relative to the elongated conformation as n increases. For n = 4, a predominantly compact globular conformation was observed. Solutions containing the peptide CaMKII(290-309), the CaM target domain of the Ca(2+)/calmodulin-dependent protein kinase II (CaMKII) enzyme, yielded predominantly globular Ca(4)(2+)-CaM-CaMKII(290-309) complexes. Similar results were obtained with the 26-residue peptide melittin. For the 14-residue C-terminal melittin fragment, on the other hand, formation of both a 1:1 and a 1:2 CaM-peptide complex was detected. On the basis of the entirety of our results, we conclude that the collapse of extended (dumbbell-like) CaM structures into more compact globular structures occurs upon specific binding of four calcium ions. Furthermore, this calcium-induced structural collapse of CaM appears to be a prerequisite for formation of a particularly stable CaM-peptide complex involving peptides long enough to be engaged in interactions with both lobes of CaM.
Wyttenbach
Thomas
T
Department of Chemistry and Biochemistry, University of California Santa Barbara, Santa Barbara, California 93106, USA.
Grabenauer
Megan
M
Thalassinos
Konstantinos
K
Scrivens
James H
JH
Bowers
Michael T
MT
D000595:Amino Acid Sequence; D002118:Calcium / Q000737:chemistry*; D054732:Calcium-Calmodulin-Dependent Protein Kinase Type 2 / Q000737:chemistry; D002147:Calmodulin / Q000737:chemistry* / Q000378:metabolism; D018360:Crystallography, X-Ray; D008024:Ligands; D013058:Mass Spectrometry; D008555:Melitten / Q000737:chemistry; D008969:Molecular Sequence Data; D010455:Peptides / Q000737:chemistry*; D017434:Protein Structure, Tertiary
D002147:Calmodulin; D008024:Ligands; D010455:Peptides; D008555:Melitten; D054732:Calcium-Calmodulin-Dependent Protein Kinase Type 2; D002118:Calcium