Molecular architecture of the rod domain of the Dictyostelium gelation factor (ABP120).
The Dictyostelium discoideum gelation factor is a two-chain actin-cross-linking protein that, in addition to an N-terminal actin-binding domain, has a rod domain constructed from six tandem repeats of a 100-residue motif that has an immunoglobulin fold. To define the architecture of the rod domain of gelation factor, we have expressed in E. coli a series of constructs corresponding to different numbers of gelation factor rod repeats and have characterised them by chemical crosslinking, ultracentrifugation, column chromatography, matrix-assisted laser desorption ionisation (MALDI) mass spectrometry and NMR spectroscopy. Fragments corresponding to repeats 1-6 and 5-6 dimerise, whereas repeats 1-5 and single repeats 3 and 4 are monomeric. Repeat 6 interacts weakly and was present as monomer and dimer when analysed by analytical ultracentrifugation. Proteolytic digestion of rod5-6 resulted in the generation of two polypeptides that roughly corresponded to rod5 and part of rod6. None of these polypeptides formed dimers after chemical crosslinking. Stable dimerisation therefore appears to require repeats 5 and 6. Based on these data a model of gelation factor architecture is presented. We suggest an arrangement of the chains where only the carboxy-terminal repeats interact as was observed for filamin/ABP280, the mammalian homologue of gelation factor.
Fucini
P
P
Max-Planck-Institut für Biochemie, Martinsried, FRG.
Köppel
B
B
Schleicher
M
M
Lustig
A
A
Holak
T A
TA
Müller
R
R
Stewart
M
M
Noegel
A A
AA
D000818:Animals; D002352:Carrier Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D002850:Chromatography, Gel; D002942:Circular Dichroism; D003432:Cross-Linking Reagents; D004023:Dictyostelium / Q000737:chemistry* / Q000235:genetics; D019281:Dimerization; D004926:Escherichia coli / Q000235:genetics; D008840:Microfilament Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D008970:Molecular Weight; D019906:Nuclear Magnetic Resonance, Biomolecular; D010446:Peptide Fragments / Q000737:chemistry / Q000378:metabolism; D011485:Protein Binding; D011487:Protein Conformation; D017510:Protein Folding; D015800:Protozoan Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D020449:Repetitive Sequences, Amino Acid; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D014461:Ultracentrifugation
D002352:Carrier Proteins; D003432:Cross-Linking Reagents; D008840:Microfilament Proteins; D010446:Peptide Fragments; D015800:Protozoan Proteins; D011993:Recombinant Fusion Proteins; C060680:abpC protein, Dictyostelium