Identification of a region in segment 1 of gelsolin critical for actin binding.

Abstract

The actin severing and capping protein gelsolin contains three distinct actin binding sites. The smallest actin binding domain of approximately 15,000 Mr was originally obtained by limited proteolysis and it corresponds to the first of six repeating segments contained in the gelsolin sequence. We have expressed this domain (here termed segment 1 or N150 to define its amino acid length) in Escherichia coli, together with a series of smaller mutants truncated at either N‐ or C‐terminal ends, in an attempt to localize residues critical of actin binding. Limited truncation of segment 1 by 11 residues at its N‐terminal end has no observable effect on actin binding, but on removal of a further eight residues, actin binding is totally eliminated. Although this loss of actin binding may reflect ablation of critical residues, we cannot rule out the possibility that removal of these residues adversely affects the folding of the polypeptide chain during renaturation. Truncation at the C‐terminus of segment 1 has a progressive effect on actin binding. Unlike intact segment 1, which shows no calcium sensitivity of actin binding within the resolution of our assays, a mutant with 19 residues deleted from its C‐terminus shows unchanged affinity for actin in the presence of calcium, but approximately 100‐fold weaker binding in its absence. Removal of an additional five residues from the C‐terminus produces a mutant that binds actin only in calcium. Further limited truncation results in progressively weaker calcium dependent binding and all binding is eliminated when a total of 29 residues has been removed. Although none of the expressed proteins on their own binds calcium, 45Ca is trapped in the complexes, including the complex between actin and segment 1 itself. These results highlight a region close to the C‐terminus of segment 1 that is essential for actin binding and demonstrate that calcium plays an important role in the high affinity actin binding by this domain of gelsolin.

Global Shapes of F-actin Depolymerization-competent Minimal Gelsolins

“…Although GSN is unable to bind F-actin in the absence of calcium, smaller variants (1-161 and 25-161) have been shown to bind F-actin in the absence of calcium, although the positioning/orientation of G1 is not optimal for actin depolymerization (18). The binding to calcium then enhances the affinity of G1 for actin by 1000-fold and facilitates its depolymerizing function (41). Thus, the calcium requirement of 1-161 and smaller fragments, for the F-actin depolymerization function, seems to be for aiding their optimal binding to actin.…”

“…Thus, these results further supported that the shift in Cc of actin in the presence of full-length GSN, G1-G3, G4-G6 and G1-G2 might be due to the barbed end capping of the actin filaments by these gelsolin truncates. However, because both the F-actin depolymerization-efficient (1-161 and 28 -161) as well as depolymerization-deficient (42-161) mutants of G1 domain were incapable of preventing the actin polymerization even at 4 nM concentration (molar ratio of 1:250 (gelsolin mutant/actin)), the small shift in the Cc of actin observed in the presence of these mutants at 1:1 to 1:8 (gelsolin mutant/actin) ratios could be due to the nonpolymerizing sequestration of the monomeric actin by these fragments (41). Likewise, at these higher molar ratios (1:1-1:8) at least a partial contribution from the G-actin sequestration in the observed Cc shift by the capping efficient gelsolin truncates also cannot be ruled out.…”

Structure of an F-actin Trimer Disrupted by Gelsolin and Implications for the Mechanism of Severing

“…Recombinant techniques permit purification of homogeneous preparations of specific segments of gelsolin where contamination with full-length gelsolin is not possible. Because the actin monomer binding activity of recombinant GS-1 has been studied extensively (3,22), in our experiments on preparation of F-actin fragments (Fig. 1), recombinant GS-1 was expected to facilitate depolymerization by both sequestering monomeric G-actin and binding to the fast growing barbed end of newly formed F-actin fragments.…”

“…To block assembly of the purified actin oligomers, we used recombinant segment-1 of gelsolin (GS-1) (21)(22)(23)(24) to generate GS-1-bound cross-linked actin oligomers. Because of its strong actin monomer binding activity (K d ϭ 5 nM) (21)(22)(23)(24), GS-1 was expected to facilitate depolymerization by both sequestering monomeric G-actin and binding to the fast growing barbed end of newly formed F-actin fragments.…”

“…Because of its strong actin monomer binding activity (K d ϭ 5 nM) (21)(22)(23)(24), GS-1 was expected to facilitate depolymerization by both sequestering monomeric G-actin and binding to the fast growing barbed end of newly formed F-actin fragments. Here, we show that mixtures of the cross-linked GS-1-bound actin oligomers can be fractionated readily according to their size and crystallized.…”