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Alternate Binding of Actin and Calmodulin to Multiple Sites on Dystrophin

1995; Elsevier BV; Volume: 270; Issue: 10 Linguagem: Inglês

10.1074/jbc.270.10.5578

1083-351X

Harry W. Jarrett, John L. Foster,

Silk-based biomaterials and applications

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.