An alpha-actinin-profilin chimaera with two alternatively operating actin-binding sites.
Studying the mode of interaction between actin and actin-binding proteins, we constructed a chimaeric protein consisting of the sequence for bovine profilin I (P), to which the sequence for the actin-binding domain of Dictyostelium discoideum alpha-actinin (alphaA1-2) was fused N-terminally. The resulting hybrid clone was expressed in Escherichia coli, and the chimaeric protein, alphaA1-2P, purified by affinity chromatography on poly-(L-proline) (PLP) columns and identified using specific antibodies. High resolution electron microscopy demonstrated that this protein consists of two discrete subdomains. In biochemical, viscometric and electron microscopic analyses, we showed that both modules in this molecule are biologically active. The chimaera binds to poly-(L-proline) and inhibits the polymerization of G-actin in KCl, which is consistent with the assumption that the profilin part is intact. Inhibition of actin polymerization in KCl was stronger than that of the parental profilin, and the Kd value of its interaction with rabbit skeletal muscle actin, as determined by falling ball viscometry, was smaller (mean value 0.5 x 10(-6) M, as compared to 1.9 x 10(-6) M for bovine profilin). In 2mM MgCl2, the actin polymerized rapidly, consistent with the interpretation that under these conditions the chimaera, like profilin, is less efficient as an actin-sequestering agent. In the presence of alphaA1-2P, the resulting filaments were decorated with particles projecting from the filament axis. We conclude that under these conditions the alphaA1-2 domain of alphaA1-2P is preferentially active, attaching the chimaeric particles laterally to the filaments. Hence, the parental modules combined in alphaA1-2P permit this molecule to switch from a G-actin- to an F-actin-binding form.
Schlüter
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Cell Biology, Zoological Institute, Technical University of Braunschweig, Germany.
Zeuschner
D
D
Winkler
J
J
Pope
B
B
Weeds
A
A
Jockusch
B M
BM
D000185:Actinin / Q000235:genetics / Q000378:metabolism* / Q000648:ultrastructure; D000199:Actins / Q000378:metabolism*; D000818:Animals; D001665:Binding Sites; D002417:Cattle; D003285:Contractile Proteins; D008274:Magnesium; D008840:Microfilament Proteins / Q000235:genetics / Q000378:metabolism* / Q000648:ultrastructure; D010455:Peptides / Q000378:metabolism; D051304:Profilins; D011817:Rabbits; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism / Q000648:ultrastructure
D000199:Actins; D003285:Contractile Proteins; D008840:Microfilament Proteins; D010455:Peptides; D051304:Profilins; D011993:Recombinant Fusion Proteins; D000185:Actinin; C011083:polyproline; D008274:Magnesium