Details

Autor(en) / Beteiligte

• HUBER, Pia A. J.
• EL-MEZGUELDI, Mohammed
• GRABAREK, Zenon
• SLATTER, David A.
• LEVINE, Barry A.
• MARSTON, Steven B.

Titel

Multiple-sited interaction of caldesmon with Ca2+-calmodulin

Ist Teil von

• Biochemical journal, 1996-06, Vol.316 (2), p.413-420

Erscheinungsjahr

1996

Quelle

EZB Electronic Journals Library

Beschreibungen/Notizen

• The binding of Ca2+– and Ba2+–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 Ca2+–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 Ca2+–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. Ca2+–calmodulin F92A produced a change in wavelength of 4 nm but no change in maximum, whereas Ca2+–calmodulin C41/75 binding produced a decrease in fluorescence with no shift of the maximum. We conclude that functional binding of Ca2+–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.