ジャーナル: Nat Commun / 年: 2020 タイトル: Structural basis for itraconazole-mediated NPC1 inhibition. 著者: Tao Long / Xiaofeng Qi / Abdirahman Hassan / Qiren Liang / Jef K De Brabander / Xiaochun Li / 要旨: Niemann-Pick C1 (NPC1), a lysosomal protein of 13 transmembrane helices (TMs) and three lumenal domains, exports low-density-lipoprotein (LDL)-derived cholesterol from lysosomes. TMs 3-7 of NPC1 ...Niemann-Pick C1 (NPC1), a lysosomal protein of 13 transmembrane helices (TMs) and three lumenal domains, exports low-density-lipoprotein (LDL)-derived cholesterol from lysosomes. TMs 3-7 of NPC1 comprise the Sterol-Sensing Domain (SSD). Previous studies suggest that mutation of the NPC1-SSD or the addition of the anti-fungal drug itraconazole abolishes NPC1 activity in cells. However, the itraconazole binding site and the mechanism of NPC1-mediated cholesterol transport remain unknown. Here, we report a cryo-EM structure of human NPC1 bound to itraconazole, which reveals how this binding site in the center of NPC1 blocks a putative lumenal tunnel linked to the SSD. Functional assays confirm that blocking this tunnel abolishes NPC1-mediated cholesterol egress. Intriguingly, the palmitate anchor of Hedgehog occupies a similar site in the homologous tunnel of Patched, suggesting a conserved mechanism for sterol transport in this family of proteins and establishing a central function of their SSDs.
電子線照射量: 80 e/Å2 / フィルム・検出器のモデル: GATAN K3 (6k x 4k)
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解析
ソフトウェア
名称: REFMAC / バージョン: 5.8.0135 / 分類: 精密化
CTF補正
タイプ: PHASE FLIPPING AND AMPLITUDE CORRECTION
3次元再構成
解像度: 4.02 Å / 解像度の算出法: FSC 0.143 CUT-OFF / 粒子像の数: 209612 / 対称性のタイプ: POINT
精密化
解像度: 4.13→231 Å / Cor.coef. Fo:Fc: 0.832 / SU B: 59.618 / SU ML: 0.778 / ESU R: 0.305 / 立体化学のターゲット値: MAXIMUM LIKELIHOOD / 詳細: HYDROGENS HAVE BEEN ADDED IN THE RIDING POSITIONS
Rfactor
反射数
%反射
Rwork
0.44541
-
-
obs
0.44541
367648
100 %
溶媒の処理
イオンプローブ半径: 0.8 Å / 減衰半径: 0.8 Å / VDWプローブ半径: 1.2 Å / 溶媒モデル: MASK