Structural basis for itraconazole-mediated NPC1 inhibition

Area of Science:

• Biochemistry
• Cell Biology
• Structural Biology

Background:

• Niemann-Pick C1 (NPC1) is a lysosomal protein crucial for cholesterol export.
• The Sterol-Sensing Domain (SSD) within NPC1 is implicated in its function, but its precise role and drug-binding sites remain unclear.
• Previous research indicates mutations in the NPC1-SSD or treatment with itraconazole impair NPC1 activity.

Purpose of the Study:

• To elucidate the binding site of itraconazole within NPC1.
• To determine the mechanism by which NPC1 mediates cholesterol transport.
• To investigate the structural basis for NPC1 function and its relation to other sterol-transporting proteins.

Main Methods:

• Cryo-electron microscopy (cryo-EM) was used to determine the structure of human NPC1 bound to itraconazole.
• Functional assays were performed to assess the impact of the identified binding site on NPC1 activity.
• Structural comparisons were made with homologous proteins, such as Patched.

Main Results:

• The cryo-EM structure revealed itraconazole binding within a central cavity of NPC1, effectively blocking a putative lumenal tunnel connected to the SSD.
• Functional assays confirmed that obstructing this tunnel significantly inhibits NPC1-mediated cholesterol egress from lysosomes.
• A conserved sterol-binding site was identified, similar to that found in the Patched protein, suggesting a shared transport mechanism.

Conclusions:

• The study identifies a critical itraconazole-binding site within NPC1 that directly blocks a lumenal tunnel essential for cholesterol transport.
• This finding establishes a functional role for this tunnel and the SSD in mediating cholesterol egress.
• A conserved sterol transport mechanism involving the SSD is proposed across related protein families.