Defining of the minimal domain of protein 4.1 involved in spectrin-actin binding.
The spectrin-actin-binding domain of protein 4.1 is encoded by a 21-amino acid alternative exon and a 59-amino acid constitutive exon. To characterize the minimal domain active for interactions with spectrin and actin, we functionally characterized recombinant 4.1 peptides containing the 21-amino acid cassette plus varying portions of the 59-amino acid cassette (designated 21.10 to 21.59). Peptide 21.43 was shown fully functional in binary interactions with spectrin (by cosedimentation and coimmunoprecipitation experiments) and in ternary complex formation with spectrin and actin (by an in vitro gelation assay). Further truncation produced peptides incapable of binary interactions but fully competent for ternary complex formation (peptides 21.36 and 21.31), shorter peptides with reduced ternary complex activity and altered kinetics (21.26 and 0.59), and inactive peptides (21.20 and 21.10). Binding studies and circular dichroism experiments suggested that residues 37-43 of the constitutive domain were directly involved in spectrin binding. These data indicate that 4.1-spectrin binary interaction requires the 21-amino acid alternative cassette plus the 43 N-terminal residues of the constitutive domain. Moreover, the existence of two possible ternary complex assembly pathways is suggested: one initiated by 4.1-spectrin interactions, and a second by 4.1-actin interactions. The latter may require a putative actin binding motif within the 26 N-terminal residues of the constitutive domain.
Schischmanoff
P O
PO
Life Sciences Division, Lawrence Berkeley Laboratory, University of California, Berkeley 94720, USA.
Winardi
R
R
Discher
D E
DE
Parra
M K
MK
Bicknese
S E
SE
Witkowska
H E
HE
Conboy
J G
JG
Mohandas
N
N
D000199:Actins / Q000378:metabolism*; D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D001665:Binding Sites; D003001:Cloning, Molecular; D003598:Cytoskeletal Proteins; D017931:DNA Primers; D004926:Escherichia coli / Q000235:genetics; D006801:Humans; D008565:Membrane Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D008969:Molecular Sequence Data; D009479:Neuropeptides; D010449:Peptide Mapping; D017433:Protein Structure, Secondary; D011817:Rabbits; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D013049:Spectrin / Q000378:metabolism*
D000199:Actins; D003598:Cytoskeletal Proteins; D017931:DNA Primers; D008565:Membrane Proteins; D009479:Neuropeptides; D011994:Recombinant Proteins; C028841:erythrocyte membrane band 4.1 protein; C120557:erythrocyte membrane protein band 4.1-like 1; D013049:Spectrin