National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)
R01GM127440, R01GM092917, S10OD012272
米国
引用
ジャーナル: Proc Natl Acad Sci U S A / 年: 2022 タイトル: Structure of Arp2/3 complex at a branched actin filament junction resolved by single-particle cryo-electron microscopy. 著者: Bojian Ding / Heidy Y Narvaez-Ortiz / Yuvraj Singh / Glen M Hocky / Saikat Chowdhury / Brad J Nolen / 要旨: Arp2/3 complex nucleates branched actin filaments that provide pushing forces to drive cellular processes such as lamellipodial protrusion and endocytosis. Arp2/3 complex is intrinsically inactive, ...Arp2/3 complex nucleates branched actin filaments that provide pushing forces to drive cellular processes such as lamellipodial protrusion and endocytosis. Arp2/3 complex is intrinsically inactive, and multiple classes of nucleation promoting factors (NPFs) stimulate its nucleation activity. When activated by WASP family NPFs, the complex must bind to the side of a preexisting (mother) filament of actin to complete the nucleation process, ensuring that WASP-mediated activation creates branched rather than linear actin filaments. How actin filaments contribute to activation is currently not understood, largely due to the lack of high-resolution structures of activated Arp2/3 complex bound to the side of a filament. Here, we present the 3.9-Å cryo-electron microscopy structure of the Arp2/3 complex at a branch junction. The structure reveals contacts between Arp2/3 complex and the side of the mother actin filament that likely stimulate subunit flattening, a conformational change that allows the actin-related protein subunits in the complex (Arp2 and Arp3) to mimic filamentous actin subunits. In contrast, limited contact between the bottom half of the complex and the mother filament suggests that clamp twisting, a second major conformational change observed in the active state, is not stimulated by actin filaments, potentially explaining why actin filaments are required but insufficient to trigger nucleation during WASP-mediated activation. Along with biochemical and live-cell imaging data and molecular dynamics simulations, the structure reveals features critical for the interaction of Arp2/3 complex with actin filaments and regulated assembly of branched actin filament networks in cells.
名称: Actin-related protein 3 / タイプ: protein_or_peptide / ID: 1 詳細: Missing sequences correspond to unmodeled regions due to poor density map コピー数: 1 / 光学異性体: LEVO
名称: Actin-related protein 2 / タイプ: protein_or_peptide / ID: 2 詳細: Missing sequences correspond to unmodelled regions due to poor density map コピー数: 1 / 光学異性体: LEVO
分子 #3: Actin-related protein 2/3 complex subunit 1B
分子
名称: Actin-related protein 2/3 complex subunit 1B / タイプ: protein_or_peptide / ID: 3 詳細: Missing sequences correspond to unmodeled regions due to poor density map コピー数: 1 / 光学異性体: LEVO
分子 #4: Actin-related protein 2/3 complex subunit 2
分子
名称: Actin-related protein 2/3 complex subunit 2 / タイプ: protein_or_peptide / ID: 4 詳細: Missing sequences correspond to unmodeled regions due to poor density map コピー数: 1 / 光学異性体: LEVO
分子 #5: Actin-related protein 2/3 complex subunit 3
分子
名称: Actin-related protein 2/3 complex subunit 3 / タイプ: protein_or_peptide / ID: 5 詳細: Missing sequences correspond to unmodeled regions due to poor density map コピー数: 1 / 光学異性体: LEVO
分子 #7: Actin-related protein 2/3 complex subunit 5
分子
名称: Actin-related protein 2/3 complex subunit 5 / タイプ: protein_or_peptide / ID: 7 詳細: Missing sequences correspond to unmodeled regions due to poor density map コピー数: 1 / 光学異性体: LEVO
名称: Actin, alpha skeletal muscle / タイプ: protein_or_peptide / ID: 8 詳細: Missing sequences correspond to unmodeled regions due to poor density map. Actin subunits corresponding to chains J,K,L,M,T and U have been trimmed to c-beta due to lack of density for ...詳細: Missing sequences correspond to unmodeled regions due to poor density map. Actin subunits corresponding to chains J,K,L,M,T and U have been trimmed to c-beta due to lack of density for modeling sidechains. These subunits were rigid body fitted into the map. コピー数: 14 / 光学異性体: LEVO
名称: Phalloidin / タイプ: protein_or_peptide / ID: 9 詳細: Phalloidin from Amanita phalloides is a rigid bicyclic heptapeptide. This is a small molecule and does not have conventional planar peptide bonds present in proteins. コピー数: 15 / 光学異性体: LEVO
Data were collected by shifting of stage to targeted exposure position. Stage was tilted to different angles: including 40 degree, 36 degree, 30 degree, 25 degree, 15 degree, 0 degree, -20 degree, and -33 degree during data collection.
撮影
フィルム・検出器のモデル: FEI FALCON III (4k x 4k) 検出モード: COUNTING / デジタル化 - サイズ - 横: 4096 pixel / デジタル化 - サイズ - 縦: 4096 pixel / デジタル化 - サンプリング間隔: 14.0 µm / 撮影したグリッド数: 3 / 実像数: 7436 / 平均露光時間: 70.0 sec. / 平均電子線量: 41.97 e/Å2 詳細: Each micrograph was acquired as dose-fractionated movies consisting of 82 frames per movie.