Characterization of the calmodulin-binding site of nonerythroid alpha-spectrin. Recombinant protein and model peptide studies.
An important function of the mammalian nonerythroid alpha-spectrin chain (alpha-fodrin) that distinguishes it from the closely related erythroid isoform is its ability to bind calmodulin. By analysis of a series of deleted recombinant spectrin fusion proteins, we have identified a region in the nonerythroid alpha chain involved in calcium-dependent binding of calmodulin. The region is distinctive in that the sequence is absent from the homologous domain of the erythroid alpha chain and diverges from the normal internal repeat structure observed throughout other spectrins. In order to determine limits of this functional site, a synthetic peptide as small as 24 residues was shown to compete with either recombinant or brain alpha-spectrin in binding to calmodulin. The active peptide, which was derived from a segment between repeats 11 and 12, was composed of the following sequence: Lys-Thr-Ala-Ser-Pro-Trp-Lys-Ser-Ala-Arg-Leu-Met-Val-His-Thr-Val-Ala-Thr-Phe-Asn - Ser-Ile-Lys-Glu. Comparison of this sequence with functional sites in other diverse calcium-dependent calmodulin-binding proteins has revealed a structural motif common to all of these proteins, namely clusters of hydrophobic residues interspersed with basic residues. When folded into alpha-helical conformations, these binding sites are predicted to form amphipathic structures.
Leto
T L
TL
Laboratory of Clinical Investigation National Institute of Allergy and Infectious Diseases, Bethesda, Maryland 20892.
Pleasic
S
S
Forget
B G
BG
Benz
E J
EJ
Marchesi
V T
VT
D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D001921:Brain / Q000378:metabolism; D002147:Calmodulin / Q000235:genetics / Q000378:metabolism*; D002352:Carrier Proteins / Q000378:metabolism*; D004247:DNA / Q000235:genetics; D004912:Erythrocytes / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics; D005822:Genetic Vectors; D008840:Microfilament Proteins / Q000378:metabolism*; D008969:Molecular Sequence Data; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D011994:Recombinant Proteins / Q000378:metabolism; D013049:Spectrin / Q000235:genetics / Q000378:metabolism*
D002147:Calmodulin; D002352:Carrier Proteins; D004247:DNA; D008840:Microfilament Proteins; D011993:Recombinant Fusion Proteins; D011994:Recombinant Proteins; D013049:Spectrin