ジャーナル: J Virol / 年: 2014 タイトル: Structure of 2G12 Fab2 in complex with soluble and fully glycosylated HIV-1 Env by negative-stain single-particle electron microscopy. 著者: Charles D Murin / Jean-Philippe Julien / Devin Sok / Robyn L Stanfield / Reza Khayat / Albert Cupo / John P Moore / Dennis R Burton / Ian A Wilson / Andrew B Ward / 要旨: The neutralizing anti-HIV-1 antibody 2G12 is of particular interest due to the sterilizing protection it provides from viral challenge in animal models. 2G12 is a unique, domain-exchanged antibody ...The neutralizing anti-HIV-1 antibody 2G12 is of particular interest due to the sterilizing protection it provides from viral challenge in animal models. 2G12 is a unique, domain-exchanged antibody that binds exclusively to conserved N-linked glycans that form the high-mannose patch on the gp120 outer domain centered on a glycan at position N332. Several glycans in and around the 2G12 epitope have been shown to interact with other potent, broadly neutralizing antibodies; therefore, this region constitutes a supersite of vulnerability on gp120. While crystal structures of 2G12 and 2G12 bound to high-mannose glycans have been solved, no structural information that describes the interaction of 2G12 with gp120 or the Env trimer is available. Here, we present a negative-stain single-particle electron microscopy reconstruction of 2G12 Fab2 in complex with a soluble, trimeric Env at ∼17-Å resolution that reveals the antibody's interaction with its native and fully glycosylated epitope. We also mapped relevant glycans in this epitope by fitting high-resolution crystal structures and by performing neutralization assays of glycan knockouts. In addition, a reconstruction at ∼26 Å of the ternary complex formed by 2G12 Fab2, soluble CD4, and Env indicates that 2G12 may block membrane fusion by induced steric hindrance upon primary receptor binding, thereby abrogating Env's interaction with coreceptor(s). These structures provide a basis for understanding 2G12 binding and neutralization, and our low-resolution model and glycan assignments provide a basis for higher-resolution studies to determine the molecular nature of the 2G12 epitope. IMPORTANCE: HIV-1 is a human virus that results in the deaths of millions of people around the world each year. While there are several effective therapeutics available to prolong life, a vaccine is ...IMPORTANCE: HIV-1 is a human virus that results in the deaths of millions of people around the world each year. While there are several effective therapeutics available to prolong life, a vaccine is the best long-term solution for curbing this global epidemic. Here, we present structural data that reveal the viral binding site of one of the first HIV-1-neutralizing antibodies isolated, 2G12, and provide a rationale for its effectiveness. These structures provide a basis for higher-resolution studies to determine the molecular nature of the 2G12 epitope, which will aid in vaccine design and antibody-based therapies.
ダウンロード / ファイル: emd_5983.map.gz / 形式: CCP4 / 大きさ: 3.3 MB / タイプ: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
注釈
Reconstruction of HIV-1 Env SOSIP BG505.664 bound to soluble, two-domain CD4 (domains 1 and 2, sCD4) and 2G12 Fabs
ボクセルのサイズ
X=Y=Z: 4.35 Å
密度
表面レベル
登録者による: 4.1 / ムービー #1: 4.1
最小 - 最大
-3.92196393 - 13.6949892
平均 (標準偏差)
0.00000001 (±0.9999994)
対称性
空間群: 1
詳細
EMDB XML:
マップ形状
Axis order
X
Y
Z
Origin
-48
-48
-48
サイズ
96
96
96
Spacing
96
96
96
セル
A=B=C: 417.59998 Å α=β=γ: 90.0 °
CCP4マップ ヘッダ情報:
mode
Image stored as Reals
Å/pix. X/Y/Z
4.35
4.35
4.35
M x/y/z
96
96
96
origin x/y/z
0.000
0.000
0.000
length x/y/z
417.600
417.600
417.600
α/β/γ
90.000
90.000
90.000
start NX/NY/NZ
-80
0
-4
NX/NY/NZ
161
13
58
MAP C/R/S
1
2
3
start NC/NR/NS
-48
-48
-48
NC/NR/NS
96
96
96
D min/max/mean
-3.922
13.695
0.000
-
添付データ
-
試料の構成要素
-
全体 : Fab fragment of 2G12 monoclonal antibody and soluble, two-domain ...
全体
名称: Fab fragment of 2G12 monoclonal antibody and soluble, two-domain CD4 (domains 1 and 2, sCD4) bound to HIV-1 Env BG505.664
要素
試料: Fab fragment of 2G12 monoclonal antibody and soluble, two-domain CD4 (domains 1 and 2, sCD4) bound to HIV-1 Env BG505.664
タンパク質・ペプチド: HIV-1 Env
タンパク質・ペプチド: soluble CD4
タンパク質・ペプチド: Human Monoclonal Antibody 2G12 IgG1 Fab Fragment
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超分子 #1000: Fab fragment of 2G12 monoclonal antibody and soluble, two-domain ...
超分子
名称: Fab fragment of 2G12 monoclonal antibody and soluble, two-domain CD4 (domains 1 and 2, sCD4) bound to HIV-1 Env BG505.664 タイプ: sample / ID: 1000 集合状態: Env trimer bound to three 2G12 domain-swapped Fabs and three sCD4s Number unique components: 3
The particles were selected using automatic selection program Leginon and processed using the Appion system.
最終 再構成
アルゴリズム: OTHER / 解像度のタイプ: BY AUTHOR / 解像度: 26.0 Å / 解像度の算出法: OTHER / ソフトウェア - 名称: EMAN2 / 詳細: Final map was low pass filtered to 20 Angstrom. / 使用した粒子像数: 5372
2G12 Fab structures were fit by rigid body fitting using the UCSF Chimera volume fit option, simulating a map at an estimated resolution of 20 Angstrom.