TY - JOUR
T1 - Structural basis for itraconazole-mediated NPC1 inhibition
AU - Long, Tao
AU - Qi, Xiaofeng
AU - Hassan, Abdirahman
AU - Liang, Qiren
AU - De Brabander, Jef K.
AU - Li, Xiaochun
N1 - Funding Information:
We thank M. Brown and J. Goldstein for their invaluable support and discussion throughout the project. The data were collected at the UT Southwestern Medical Center Cryo-EM Facility (funded in part by the CPRIT Core Facility Support Award RP170644). We thank our colleague D. Stoddard for assistance in data collection, F. Lu and M. Trinh for assistance in NPC1 functional assays, J. Seemann for assistance in immunofluorescence microscopy, L. Beatty, P. Schmiege, and R. Wang for technical help, and E. Coutavas and E. Debler for discussion during paper preparation. This work was supported by NIH grant P01 HL020948, R01 GM134700 and R01 GM135343, the Endowed Scholars Program in Medical Science of UT Southwestern Medical Center, O’Donnell Junior Faculty Funds, Welch Foundation (I-1957) (to X.L.) and Welch Foundation (I-1422) (to J.K.D.B.). J.K.D.B. holds the Julie and Louis Beecherl, Jr., Chair in Medical Science at UT Southwestern Medical Center. X.Q. is the recipient of a DDBrown Fellow of the Life Sciences Research Foundation. X.L. is a Damon Runyon-Rachleff Innovator supported by the Damon Runyon Cancer Research Foundation (DRR-53-19) and a Rita C. and William P. Clements Jr. Scholar in Biomedical Research at UT Southwestern Medical Center.
Publisher Copyright:
© 2020, The Author(s).
PY - 2020/12/1
Y1 - 2020/12/1
N2 - 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.
AB - 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.
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U2 - 10.1038/s41467-019-13917-5
DO - 10.1038/s41467-019-13917-5
M3 - Article
C2 - 31919352
AN - SCOPUS:85077709950
SN - 2041-1723
VL - 11
JO - Nature communications
JF - Nature communications
IS - 1
M1 - 152
ER -