ジャーナル: PLoS Pathog / 年: 2020 タイトル: Cryo-EM structure of the fully-loaded asymmetric anthrax lethal toxin in its heptameric pre-pore state. 著者: Claudia Antoni / Dennis Quentin / Alexander E Lang / Klaus Aktories / Christos Gatsogiannis / Stefan Raunser / 要旨: Anthrax toxin is the major virulence factor secreted by Bacillus anthracis, causing high mortality in humans and other mammals. It consists of a membrane translocase, known as protective antigen (PA) ...Anthrax toxin is the major virulence factor secreted by Bacillus anthracis, causing high mortality in humans and other mammals. It consists of a membrane translocase, known as protective antigen (PA), that catalyzes the unfolding of its cytotoxic substrates lethal factor (LF) and edema factor (EF), followed by translocation into the host cell. Substrate recruitment to the heptameric PA pre-pore and subsequent translocation, however, are not well understood. Here, we report three high-resolution cryo-EM structures of the fully-loaded anthrax lethal toxin in its heptameric pre-pore state, which differ in the position and conformation of LFs. The structures reveal that three LFs interact with the heptameric PA and upon binding change their conformation to form a continuous chain of head-to-tail interactions. As a result of the underlying symmetry mismatch, one LF binding site in PA remains unoccupied. Whereas one LF directly interacts with a part of PA called α-clamp, the others do not interact with this region, indicating an intermediate state between toxin assembly and translocation. Interestingly, the interaction of the N-terminal domain with the α-clamp correlates with a higher flexibility in the C-terminal domain of the protein. Based on our data, we propose a model for toxin assembly, in which the relative position of the N-terminal α-helices in the three LFs determines which factor is translocated first.
Fully-loaded anthrax lethal toxin in its heptameric pre-pore state and PA7LF(2+1A) arrangement
COMPLEX
Heptameric pre-pores of proteolytically-acitivated protective antigen were loaded with excess of LFs to create the PA7LF3 complexes. The LFs form a continuous chain of head-to-tail interactions. In the PA7LF(2+1A) arrangement, the third LF binds with its N-terminal domain to the C-terminal region of the 2nd LF.
all
0
MULTIPLESOURCES
2
Protectiveantigen
COMPLEX
The trypsin-activated 63 kDa fragments assemble into a hepatameric pre-pore
#1
1
RECOMBINANT
3
Lethalfactor
COMPLEX
Three LF molecules crown the heptameric PA ring. The LFs form a continuous chain of head-to-tail interactions. In the PA7LF(2+1A) arrangement, the third LF binds with its N-terminal domain to the C-terminus of the 2nd LF.
#2
1
RECOMBINANT
分子量
ID
Entity assembly-ID
値 (°)
実験値
1
1
0.72MDa
NO
2
1
0.063MDa
NO
3
1
0.093MDa
NO
由来(天然)
ID
Entity assembly-ID
生物種
Ncbi tax-ID
2
2
Bacillus anthracis (炭疽菌)
1392
3
3
Bacillus anthracis (炭疽菌)
1392
由来(組換発現)
ID
Entity assembly-ID
生物種
Ncbi tax-ID
2
2
Escherichia coli BL21(DE3) (大腸菌)
469008
3
3
Bacillus anthracis (炭疽菌)
1392
緩衝液
pH: 8.5
緩衝液成分
ID
濃度
名称
式
Buffer-ID
1
20mM
Trishydrochloride
Tris-HCl
1
2
150mM
sodiumchloride
NaCl
1
試料
濃度: 0.06 mg/ml / 包埋: NO / シャドウイング: NO / 染色: NO / 凍結: YES
平均露光時間: 15 sec. / 電子線照射量: 74.4 e/Å2 / 検出モード: COUNTING フィルム・検出器のモデル: GATAN K2 SUMMIT (4k x 4k) 実像数: 5238
電子光学装置
エネルギーフィルター名称: GIF Bioquantum / エネルギーフィルタースリット幅: 20 eV
画像スキャン
動画フレーム数/画像: 40
-
解析
ソフトウェア
名称: PHENIX / バージョン: 1.18.2_3874: / 分類: 精密化
EMソフトウェア
ID
名称
バージョン
カテゴリ
1
crYOLO
粒子像選択
2
EPU
1.1
画像取得
4
SPHIRE
CTF補正
10
SPHIRE
最終オイラー角割当
11
SPHIRE
分類
12
SPHIRE
3次元再構成
13
PHENIX
1.18.2
モデル精密化
14
Coot
0.8.9.1
モデル精密化
CTF補正
タイプ: PHASE FLIPPING AND AMPLITUDE CORRECTION
粒子像の選択
選択した粒子像数: 382000
対称性
点対称性: C1 (非対称)
3次元再構成
解像度: 4.2 Å / 解像度の算出法: FSC 0.143 CUT-OFF / 粒子像の数: 44000 / アルゴリズム: BACK PROJECTION / 対称性のタイプ: POINT
原子モデル構築
詳細: The previously generated PA7LF(2+1B) model (PDB:6ZXK) served as starting point and was placed into the density using rigid-body fit in Chimera. From this model, chain J was fitted into the ...詳細: The previously generated PA7LF(2+1B) model (PDB:6ZXK) served as starting point and was placed into the density using rigid-body fit in Chimera. From this model, chain J was fitted into the density corresponding to the third LF, located adjacent to the second LF. For the entire model a restrained refinement in phenix was performed. The resulting model was further refined with a combination of phenix and coot. Unresolved regions were deleted and side chain information was removed for less well-resolved regions.