Functional characterization of recombinant human red cell alpha-spectrin polypeptides containing the tetramer binding site.
Spectrin, a heterodimer composed of alpha and beta subunits, interacts with itself head-to-head to form tetramers in the erythrocyte membrane cytoskeleton. The NH2-terminal region of alpha-spectrin, encompassing the alpha I 80-kDa domain, was expressed in Escherichia coli. In addition to the correctly initiated polypeptide, four smaller polypeptides were produced by initiation at internal codons. Only the full-length polypeptide was able to bind to spectrin dimers, beta monomers, and to a recombinant polypeptide containing the COOH terminus of beta-spectrin. The head-to-head interaction with beta-spectrin was also retained by a recombinant polypeptide containing the NH2-terminal 158 amino acids of the alpha subunit. Deletion of the first 27 or 49 NH2-terminal amino acids abolished binding of this polypeptide to the beta monomer. The phasing used to design these recombinant polypeptides was based on a conformational model recently refined by Speicher et al. (Speicher, D. W., DeSilva, T. M., Speicher, K. D., Ursitti, J. A., Hembach, P., and Weglarz, L. (1993) J. Biol. Chem. 268, 4227-4235), where the structural unit begins and terminates around residue 30 of the repeat unit. The binding properties, mobility on gel filtration, and circular dichroism data of the recombinant polypeptides indicated that most polypeptides were able to assume their native conformation.
Kotula
L
L
Wistar Institute of Anatomy and Biology, Philadelphia, Pennsylvania 19104.
DeSilva
T M
TM
Speicher
D W
DW
Curtis
P J
PJ
D000595:Amino Acid Sequence; D001483:Base Sequence; D001665:Binding Sites; D001704:Biopolymers; D004247:DNA; D004912:Erythrocytes / Q000378:metabolism*; D006801:Humans; D008969:Molecular Sequence Data; D010455:Peptides / Q000235:genetics / Q000378:metabolism; D011487:Protein Conformation; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D013049:Spectrin / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*
D001704:Biopolymers; D010455:Peptides; D011994:Recombinant Proteins; D013049:Spectrin; D004247:DNA