National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)
GM097348
米国
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)
GM110533001
米国
引用
ジャーナル: Proc Natl Acad Sci U S A / 年: 2020 タイトル: Structures of cofilin-induced structural changes reveal local and asymmetric perturbations of actin filaments. 著者: Andrew R Huehn / Jeffrey P Bibeau / Anthony C Schramm / Wenxiang Cao / Enrique M De La Cruz / Charles V Sindelar / 要旨: Members of the cofilin/ADF family of proteins sever actin filaments, increasing the number of filament ends available for polymerization or depolymerization. Cofilin binds actin filaments with ...Members of the cofilin/ADF family of proteins sever actin filaments, increasing the number of filament ends available for polymerization or depolymerization. Cofilin binds actin filaments with positive cooperativity, forming clusters of contiguously bound cofilin along the filament lattice. Filament severing occurs preferentially at boundaries between bare and cofilin-decorated (cofilactin) segments and is biased at 1 side of a cluster. A molecular understanding of cooperative binding and filament severing has been impeded by a lack of structural data describing boundaries. Here, we apply methods for analyzing filament cryo-electron microscopy (cryo-EM) data at the single subunit level to directly investigate the structure of boundaries within partially decorated cofilactin filaments. Subnanometer resolution maps of isolated, bound cofilin molecules and an actin-cofilactin boundary indicate that cofilin-induced actin conformational changes are local and limited to subunits directly contacting bound cofilin. An isolated, bound cofilin compromises longitudinal filament contacts of 1 protofilament, consistent with a single cofilin having filament-severing activity. An individual, bound phosphomimetic (S3D) cofilin with weak severing activity adopts a unique binding mode that does not perturb actin structure. Cofilin clusters disrupt both protofilaments, consistent with a higher severing activity at boundaries compared to single cofilin. Comparison of these structures indicates that this disruption is substantially greater at pointed end sides of cofilactin clusters than at the barbed end. These structures, with the distribution of bound cofilin clusters, suggest that maximum binding cooperativity is achieved when 2 cofilins occupy adjacent sites. These results reveal the structural origins of cooperative cofilin binding and actin filament severing.
フィルム・検出器のモデル: GATAN K2 SUMMIT (4k x 4k) 平均電子線量: 50.0 e/Å2
電子線
加速電圧: 200 kV / 電子線源: FIELD EMISSION GUN
電子光学系
照射モード: SPOT SCAN / 撮影モード: BRIGHT FIELD
実験機器
モデル: Tecnai F20 / 画像提供: FEI Company
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画像解析
粒子像選択
選択した数: 217500 詳細: Both bare and S3D-cofilin-decorated filament segments were selected and initially refined together.
初期モデル
モデルのタイプ: OTHER
最終 再構成
使用したクラス数: 1 / 解像度のタイプ: BY AUTHOR / 解像度: 7.5 Å / 解像度の算出法: FSC 0.143 CUT-OFF 詳細: Segments with isolated, bound S3D-cofilin were split into even and odd halves for FSC calculations. 使用した粒子像数: 20082