A cross-linked profilin-actin heterodimer interferes with elongation at the fast-growing end of F-actin.
Profilin and beta/gamma-actin from calf thymus were covalently linked using the zero-length cross-linker 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide in combination with N-hydroxysuccinimide, yielding a single product with an apparent molecular mass of 60 kDa. Sequence analysis and x-ray crystallographic investigations showed that the cross-linked residues were glutamic acid 82 of profilin and lysine 113 of actin. The cross-linked complex was shown to bind with high affinity to deoxyribonuclease I and poly(l-proline). It also bound and exchanged ATP with kinetics close to that of unmodified profilin-actin and inhibited the intrinsic ATPase activity of actin. This inhibition occurred even in conditions where actin normally forms filaments. By these criteria the cross-linked profilin-actin complex retains the characteristics of unmodified profilin-actin. However, the cross-linked complex did not form filaments nor copolymerized with unmodified actin, but did interfere with elongation of actin filaments in a concentration-dependent manner. These results support a polymerization mechanism where the profilin-actin heterodimer binds to the (+)-end of actin filaments, followed by dissociation of profilin, and ATP hydrolysis and P(i) release from the actin subunit as it assumes its stable conformation in the helical filament.
Nyman
Tomas
T
Department of Cell Biology, the Wenner-Gren Institute, Stockholm University, S-106 91 Stockholm, Sweden.
Page
Rebecca
R
Schutt
Clarence E
CE
Karlsson
Roger
R
Lindberg
Uno
U
D000199:Actins / Q000737:chemistry / Q000378:metabolism* / Q000648:ultrastructure; D000251:Adenosine Triphosphatases / Q000378:metabolism*; D000255:Adenosine Triphosphate / Q000378:metabolism; D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D002417:Cattle; D003285:Contractile Proteins / Q000737:chemistry / Q000378:metabolism / Q000648:ultrastructure; D003432:Cross-Linking Reagents; D003460:Crystallization; D019281:Dimerization; D007700:Kinetics; D008840:Microfilament Proteins / Q000737:chemistry / Q000378:metabolism* / Q000648:ultrastructure; D008854:Microscopy, Electron; D008958:Models, Molecular; D051304:Profilins; D020033:Protein Isoforms / Q000378:metabolism; D055503:Protein Multimerization / Q000502:physiology; D013950:Thymus Gland / Q000378:metabolism
D000199:Actins; D003285:Contractile Proteins; D003432:Cross-Linking Reagents; D008840:Microfilament Proteins; D051304:Profilins; D020033:Protein Isoforms; D000255:Adenosine Triphosphate; D000251:Adenosine Triphosphatases