scholarly journals The Dictyostelium gelation factor shares a putative actin binding site with alpha-actinins and dystrophin and also has a rod domain containing six 100-residue motifs that appear to have a cross-beta conformation.

1989 ◽  
Vol 109 (2) ◽  
pp. 607-618 ◽  
Author(s):  
A A Noegel ◽  
S Rapp ◽  
F Lottspeich ◽  
M Schleicher ◽  
M Stewart
Keyword(s):  
Amino Acids ◽  
Binding Site ◽  
Alpha Helix ◽  
Actin Binding ◽  
Calculated Molecular Mass ◽  
Sds Page ◽  
Helical Content ◽  
High Beta ◽  
Factor Shares ◽  
Actin Binding Site

The 120-kD gelation factor and alpha-actinin are among the most abundant F-actin cross-linking proteins in Dictyostelium discoideum. Both molecules are homodimers and have extended rod-like configurations that are respectively approximately 35 and 40 nm long. Here we report the complete cDNA sequence of the 120-kD gelation factor which codes for a protein of 857 amino acids. Its calculated molecular mass is 92.2 kD which is considerably smaller than suggested by its mobility in SDS-PAGE. Analysis of the sequence shows a region that is highly homologous to D. discoideum alpha-actinin, chicken fibroblast alpha-actinin, and human dystrophin. This conserved domain probably represents an actin binding site that is connected to the rod-forming part of the molecule via a highly charged stretch of amino acids. Whereas the sequence of alpha-actinin (Noegel, A., W. Witke, and M. Schleicher. 1987. FEBS [Fed. Eur. Biochem. Soc.] Lett. 221:391-396) suggests that the extended rod domain of the molecule is based on four spectrin-like repeats with high alpha-helix potential, the rod domain of the 120-kD gelation factor is constructed from six 100-residue repeats that have a high content of glycine and proline residues and which, in contrast to alpha-actinin, do not appear to have a high alpha-helical content. These repeats show a distinctive pattern of regions that have high beta-sheet potential alternating with short zones rich in residues with a high potential for turns. This observation suggests that each 100-residue motif has a cross-beta conformation with approximately nine sheets arranged perpendicular to the long axis of the molecule. In the high beta-potential zones every second residue is often hydrophobic. In a cross-beta structure, this pattern would result in one side of the domain having a surface rich in hydrophobic side chains which could account for the dimerization of the 120-kD gelation factor subunits.

scholarly journals Domain structure in actin-binding proteins: expression and functional characterization of truncated severin.

1991 ◽  
Vol 112 (4) ◽  
pp. 665-676 ◽  
Author(s):  
L Eichinger ◽  
A A Noegel ◽  
M Schleicher
Keyword(s):  
Amino Acids ◽  
Binding Site ◽  
Binding Sites ◽  
Actin Filaments ◽  
Functional Characterization ◽  
Tryptophan Fluorescence ◽  
Border Region ◽  
Actin Binding ◽  
Capping Proteins ◽  
Actin Binding Site

Severin from Dictyostelium discoideum is a Ca2(+)-activated actin-binding protein that severs actin filaments, nucleates actin assembly, and caps the fast growing ends of actin filaments. Sequence comparison with functionally related proteins, such as gelsolin, villin, or fragmin revealed highly conserved domains which are thought to be of functional significance. To attribute the different activities of the severin molecule to defined regions, progressively truncated severin polypeptides were constructed. The complete cDNA coding for 362 (DS362) amino acids and five 3' deletions coding for 277 (DS277), 177 (DS177), 151 (DS151), 117 (DS117), or 111 (DS111) amino acids were expressed in Escherichia coli. The proteins were purified to homogeneity and then characterized with respect to their effects on the polymerization or depolymerization kinetics of G- or F-actin solutions and their binding to G-actin. Furthermore, the Ca2+ binding of these proteins was investigated with a 45Ca-overlay assay and by monitoring Ca2(+)-dependent changes in tryptophan fluorescence. Bacterially expressed DS362 showed the same Ca2(+)-dependent activities as native severin. DS277, missing the 85 COOH-terminal amino acids of severin, had lost its strict Ca2+ regulation and displayed a Ca2(+)-independent capping activity, but was still Ca2+ dependent in its severing and nucleating activities. DS151 which corresponded to the first domain of gelsolin or villin had completely lost severing and nucleating properties. However, a residual severing activity of approximately 2% was detectable if 26 amino acids more were present at the COOH-terminal end (DS177). This locates similar to gelsolin the second actin-binding site to the border region between the first and second domain. Measuring the fluorescence enhancement of pyrene-labeled G-actin in the presence of DS111 showed that the first actin-binding site was present in the NH2-terminal 111 amino acids. Extension by six or more amino acids stabilized this actin-binding site in such a way that DS117 and even more pronounced DS151 became Ca2(+)-independent capping proteins. In comparison to many reports on gelsolin we draw the following conclusions. Among the three active actin-binding sites in gelsolin the closely neighboured sites one and two share the F-actin fragmenting function, whereas the actin-binding sites two and three, which are located in far distant domains, collaborate for nucleation. In contrast, severin contains two active actin-binding sites which are next to each other and are responsible for the severing as well as the nucleating function. The single actin-binding site near the NH2-terminus is sufficient for capping of actin filaments.

scholarly journals Ezrin self-association involves binding of an N-terminal domain to a normally masked C-terminal domain that includes the F-actin binding site.

1995 ◽  
Vol 6 (8) ◽  
pp. 1061-1075 ◽  
Author(s):  
R Gary ◽  
A Bretscher
Keyword(s):  
Amino Acids ◽  
Binding Site ◽  
Sodium Dodecyl ◽  
Actin Binding ◽  
Terminal Domain ◽  
Cell Extracts ◽  
C Terminus ◽  
Self Association ◽  
Actin Binding Site

Ezrin is a membrane-cytoskeletal linking protein that is concentrated in actin-rich surface structures. It is closely related to the microvillar proteins radixin and moesin and to the tumor suppressor merlin/schwannomin. Cell extracts contain ezrin dimers and ezrin-moesin heterodimers in addition to monomers. Truncated ezrin fusion proteins were assayed by blot overlay to determine which regions mediate self-association. Here we report that ezrin self-association occurs by head-to-tail joining of distinct N-terminal and C-terminal domains. It is likely that these domains, termed N- and C-ERMADs (ezrin-radixin-moesin association domain), are responsible for homotypic and heterotypic associations among ERM family members. The N-ERMAD of ezrin resided within amino acids 1-296; deletion of 10 additional residues resulted in loss of activity. The C-ERMAD was mapped to the last 107 amino acids of ezrin, residues 479-585. The two residues at the C-terminus were required for activity, and the region from 530-585 was insufficient. The C-ERMAD was masked in the native monomer. Exposure of this domain required unfolding ezrin with sodium dodecyl sulfate or expressing the domain as part of a truncated protein. Intermolecular association could not occur unless the C-ERMAD had been made accessible to its N-terminal partner. It can be inferred that dimerization in vivo requires an activation step that exposes this masked domain. The conformationally inaccessible C-terminal region included the F-actin binding site, suggesting that this activity is likewise regulated by masking.

scholarly journals Evidence that a 27-residue sequence is the actin-binding site of ABP-120

1991 ◽  
Vol 266 (20) ◽  
pp. 12989-12993
Author(s):  
A.R. Bresnick ◽  
P.A. Janmey ◽  
J. Condeelis
Keyword(s):  
Binding Site ◽  
Actin Binding ◽  
Actin Binding Site

scholarly journals Inhibition of actin polymerization by a synthetic dodecapeptide patterned on the sequence around the actin-binding site of cofilin

1991 ◽  
Vol 266 (16) ◽  
pp. 10485-10489
Author(s):  
N. Yonezawa ◽  
E. Nishida ◽  
K. Iida ◽  
H. Kumagai ◽  
I. Yahara ◽  
...  
Keyword(s):  
Binding Site ◽  
Actin Polymerization ◽  
Actin Binding ◽  
Actin Binding Site
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