Location of two contact sites between human smooth muscle caldesmon and Ca(2+)-calmodulin.
We measured Ca(2+)-calmodulin binding to expressed human caldesmon fragments by three techniques: tryptophan fluorescence enhancement, change in fluorescence of TA-calmodulin, and cosedimentation with calmodulin-Sepharose. Ca(2+)-calmodulin bound with similar affinity to peptide M73 (C714SMWEKGNVFSSPGF727, N terminus of domain 4b), to all the fragments of caldesmon containing this peptide, and also to H9 (Thr726-Val793), which did not contain this peptide (Kd = 0.2-0.8 microM). We conclude that Ca(2+)-calmodulin binds at two sites on caldesmon; site A is the sequence 715MWEKGNVFS723 previously identified by Zhan et al. (Zhan, Q., Wong, S. S., and Wang, C.-L.A. (1991) J. Biol. Chem. 266, 21810-21814), and site B is located nearer the C terminus of caldesmon. Ca(2+)-calmodulin binding at site B is coupled to reversal of caldesmon inhibition of actin-tropomyosin activated myosin MgATPase, while calmodulin binding at site A has no detectable function. H9 did not displace M73 from Ca(2+)-calmodulin, while the other fragments did. High concentrations of M73 (> 1000 x Kd) could not displace H9 bound to Ca(2+)-calmodulin-Sepharose. Thus sites A and B in calmodulin are functionally separate. Analysis of overlapping expressed fragments indicates that site B is located in the sequence Thr726-Leu767, which includes Trp749. The minimal Ca(2+)-calmodulin binding sequence could be 744SRINEWLTK752.
Marston
S B
SB
Department of Cardiac Medicine, National Heart and Lung Institute, London, United Kingdom.
Fraser
I D
ID
Huber
P A
PA
Pritchard
K
K
Gusev
N B
NB
Torok
K
K
D000199:Actins / Q000378:metabolism; D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D001921:Brain / Q000378:metabolism; D002118:Calcium / Q000378:metabolism*; D002147:Calmodulin / Q000737:chemistry* / Q000378:metabolism*; D002148:Calmodulin-Binding Proteins / Q000737:chemistry* / Q000378:metabolism*; D002417:Cattle; D006801:Humans; D007700:Kinetics; D008969:Molecular Sequence Data; D009218:Myosins / Q000378:metabolism; D010446:Peptide Fragments / Q000138:chemical synthesis / Q000737:chemistry / Q000378:metabolism; D014335:Tropomyosin / Q000378:metabolism
D000199:Actins; D002147:Calmodulin; D002148:Calmodulin-Binding Proteins; D010446:Peptide Fragments; D014335:Tropomyosin; D009218:Myosins; D002118:Calcium