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Structural and Functional Analyses of the Gelsolin Homology Domains of Flightless‐I in Actin Dynamics
Author(s) -
Gaszler Péter,
Hanifatul Rahmah,
Pintér Réka,
Bukovics Péter,
Sakenov Rauan,
Huber Tamás,
Vig Andrea Teréz,
Tóth Mónika Ágnes,
Bugyi Beáta
Publication year - 2021
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.2021.35.s1.00391
Subject(s) - gelsolin , homology (biology) , actin , evolutionary biology , biology , computational biology , microbiology and biotechnology , chemistry , genetics , gene
Flightless‐I is a unique member of the gelsolin superfamily alloying six gelsolin homology domains and leucine‐rich repeats. Flightless‐I is an established regulator of the actin cytoskeleton, however, its biochemical activities in actin dynamics are still largely elusive. To better understand the background of the biological functioning of Flightless‐I we studied the actin activities of Drosophila Flightless‐I by in vitro bulk uorescence spectroscopy and single lament uorescence microscopy. Our work identies the rst three gelsolin homology domains (1–3) of Flightless‐I as the main actin‐binding site; neither the other three gelsolin homology domains (4–6) nor the leucine‐rich repeats bind actin. Flightless‐I inhibits polymerization by high‐afnity (∼nM) lament barbed end capping, moderately facilitates nucleation by low‐afnity (∼ µM) monomer binding, and does not sever actin laments. Our work reveals that in the presence of prolin Flightless‐I is only able to cap actin lament barbed ends but fails to promote actin assembly. Flightless‐I was found to interact with actin and affect actin dynamics in a calcium‐independent fashion in vitro, suggesting the lack of calcium‐mediated activation and conformational change of protein. For the comparative structural analysis of the six gelsolin homology domains (GH16) of gelsolin and Flightless‐I, we used a combination of biophysical and biochemical approaches. The use of both internal (tryptophans) and external (8‐anilinonaphthalene‐1‐sulfonic acid; ANS) fluorophores revealed that calcium‐binding induces structural changes in gelsolin but the conformational behavior of Flightless‐I GH16 was not significantly affected by the divalent cation. Different kinetics of limited proteolysis observed for gelsolin and Flightless‐I GH16 further strengthened this conclusion. Our experimental findings are supported by bioinformatics analysis predicting that the sequence elements responsible for Ca2+‐activation of GSN are not conserved in Flightless‐I GH16.