Alternate binding of actin and calmodulin to multiple sites on dystrophin.
Mouse dystrophin protein sequence 1-385 and various deletion mutants were expressed in Escherichia coli as fusion proteins, and the binding of actin, calmodulin, and troponin C were characterized. The fusion protein-containing sequence 1-385 bound actin with an apparent dissociation constant of 129 +/- 65 nM as measured using a solid-phase immunoassay. High affinity was also observed with ultracentrifuge cosedimentation assays and biotinylated-actin binding assays. Results with deletion mutants and analysis based upon sequence homology were consistent with two or three high affinity F-actin-binding sequences within this region of dystrophin at sequence positions 18-37 (ABS 1), 128-149 (ABS 2), and potentially at a new region called ABS 3 (86-120). A fusion protein lacking these sequences but containing dystrophin triple-helix sequences also bound actin but with reduced affinity. Calmodulin binds to dystrophin sequence 1-385 in a Ca(2+)-dependent manner and competitively inhibits F-actin binding. Results were consistent with two Ca(2+)-calmodulin-binding sites in this region of dystrophin at approximate sequence positions 18-42 (CBS 1) and 104-125 (CBS 2) with calmodulin affinities of 2.1 +/- 1 and 1.6 +/- 1.2 microM, respectively. Troponin C can substitute for calmodulin, although it binds with about 2-fold lower affinity. These results suggest that calmodulin (or troponin C) binding alternates with and may regulate F-actin binding by dystrophin much as has been postulated for other cytoskeletal proteins which are homologous to dystrophin.
Jarrett
H W
HW
Department of Biochemistry, University of Tennessee, Memphis 38163.
Foster
J L
JL
D000199:Actins / Q000378:metabolism*; D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D001665:Binding Sites; D002147:Calmodulin / Q000378:metabolism*; D003001:Cloning, Molecular; D017931:DNA Primers; D016189:Dystrophin / Q000096:biosynthesis / Q000737:chemistry / Q000378:metabolism*; D004926:Escherichia coli; D006801:Humans; D007700:Kinetics; D051379:Mice; D008969:Molecular Sequence Data; D016296:Mutagenesis; D011485:Protein Binding; D017433:Protein Structure, Secondary; D011817:Rabbits; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000378:metabolism; D017384:Sequence Deletion; D017386:Sequence Homology, Amino Acid
D000199:Actins; D002147:Calmodulin; D017931:DNA Primers; D016189:Dystrophin; D011993:Recombinant Fusion Proteins