Characterisation of the effects of mutation of the caldesmon sequence 691glu-trp-leu-thr-lys-thr696 to pro-gly-his-tyr-asn-asn on caldesmon-calmodulin interaction.
We have investigated the functional properties of a mutant (Cg1) derived from the C-terminal 99 amino acids of chicken caldesmon, 658-756 (658C) where the sequence 691glu-trp-leu-thr-lys-thr696 is changed to pro-gly-his-tyr-asn-asn. Cg1 bound Ca2+-calmodulin with (1/7)th of the affinity as compared to 658C or whole caldesmon. NMR titrations indicate that the contacts of Ca2+-calmodulin with the Trp-722 region of the peptide are retained but that those at the mutated site are lost. Most importantly Ca2+-calmodulin is not able to reverse the Cg1-induced inhibition. We conclude that the interaction of calmodulin with this caldesmon sequence is crucial for the reversal of caldesmon inhibition of actin-tropomyosin activation of myosin ATPase. The results are interpreted in terms of multisite attachment of actin and Ca2+-calmodulin to overlapping sequences in caldesmon domain 4b.
Huber
P A
PA
Imperial College School of Medicine at the National Heart and Lung Institute, Cardiac Medicine, London, UK.
Levine
B A
BA
Copeland
O
O
Marston
S B
SB
El-Mezgueldi
M
M
D000818:Animals; D017301:Ca(2+) Mg(2+)-ATPase / Q000037:antagonists & inhibitors / Q000378:metabolism; D002118:Calcium / Q000378:metabolism; D002147:Calmodulin / Q000378:metabolism*; D002148:Calmodulin-Binding Proteins / Q000235:genetics / Q000378:metabolism*; D002417:Cattle; D002645:Chickens; D004791:Enzyme Inhibitors / Q000378:metabolism*; D016296:Mutagenesis; D009218:Myosins / Q000378:metabolism; D019906:Nuclear Magnetic Resonance, Biomolecular / Q000379:methods; D011817:Rabbits; D012756:Sheep; D013329:Structure-Activity Relationship; D017186:Titrimetry
D002147:Calmodulin; D002148:Calmodulin-Binding Proteins; D004791:Enzyme Inhibitors; D017301:Ca(2+) Mg(2+)-ATPase; D009218:Myosins; D002118:Calcium