title: Multiple-sited interaction of caldesmon with Ca(2+)-calmodulin.
abstract: The binding of Ca(2+)- and Ba(2+)-calmodulin to caldesmon and its functional consequence was investigated with three different calmodulin mutants. Two calmodulin mutants have pairs of cysteine residues substituted and oxidized to a disulphide bond in either the N- or C-terminal lobe (C41/75 and C85/112). The third mutant has phenylalanine-92 replaced by alanine (F92A). Binding measurements in the presence of Ca2+ by separation on native gels and by carbodiimide-induced cross-linking showed a lower affinity for caldesmon in all the mutants. When Ca2+ was replaced by Ba2+ the affinity of calmodulin for caldesmon was further reduced. The ability of Ca(2+)-calmodulin to release caldesmon's inhibition of the actin-tropomyosin-activated myosin ATPase was virtually abolished by mutation of phenylalanine-92 to alanine or by replacing Ba2+ for Ca2+ in native calmodulin. Both cysteine mutants retained their functional ability, but the increased concentration needed for 50% release of caldesmon inhibition reflected their decreased affinity. Ca2+ -calmodulin produced a broadening in the signals of the NMR spectrum of the 10 kDa Ca(2+)-calmodulin-binding C-terminal fragment of caldesmon arising from tryptophans -749 and -779 and caused an enhancement of maximum tryptophan fluorescence of 49% and a 16 nm blue shift of the maximum. Ca(2+)-calmodulin F92A produced a change in wavelength of 4 nm but no change in maximum, whereas Ca(2+)-calmodulin C41/75 binding produced a decrease in fluorescence with no shift of the maximum. We conclude that functional binding of Ca(2+)-calmodulin to caldesmon requires multiple interaction sites on both molecules. However, some structural modification in calmodulin does not abolish the caldesmon-related functionality. This suggests that various EF hand proteins can substitute for the calmodulin molecule.
authors.affiliation: Imperial College, National Heart and Lung Institute, London, U.K.
authors.forename: P A
authors.initials: PA
authors.lastname: Huber
authors.forename: M
authors.initials: M
authors.lastname: El-Mezgueldi
authors.forename: Z
authors.initials: Z
authors.lastname: Grabarek
authors.forename: D A
authors.initials: DA
authors.lastname: Slatter
authors.forename: B A
authors.initials: BA
authors.lastname: Levine
authors.forename: S B
authors.initials: SB
authors.lastname: Marston
mesh_terms: D000251:Adenosine Triphosphatases / Q000037:antagonists & inhibitors; D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D002118:Calcium / Q000378:metabolism; D002147:Calmodulin / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D002148:Calmodulin-Binding Proteins / Q000737:chemistry / Q000378:metabolism* / Q000494:pharmacology; D002417:Cattle; D002645:Chickens; D003432:Cross-Linking Reagents / Q000378:metabolism / Q000494:pharmacology; D004591:Electrophoresis, Polyacrylamide Gel; D005022:Ethyldimethylaminopropyl Carbodiimide; D009682:Magnetic Resonance Spectroscopy; D008969:Molecular Sequence Data; D009154:Mutation; D010446:Peptide Fragments / Q000378:metabolism; D012756:Sheep; D013050:Spectrometry, Fluorescence; D013388:Succinimides / Q000378:metabolism / Q000494:pharmacology
chemical_list: D002147:Calmodulin; D002148:Calmodulin-Binding Proteins; D003432:Cross-Linking Reagents; D010446:Peptide Fragments; D013388:Succinimides; D000251:Adenosine Triphosphatases; C001426:N-hydroxysuccinimide; D005022:Ethyldimethylaminopropyl Carbodiimide; D002118:Calcium