Inter-domain dynamics drive cholesterol transport by NPC1 and NPC1L1 proteins

Area of Science:

• Biochemistry
• Molecular Biology
• Cell Biology

Background:

• Lysosomal cholesterol efflux relies on NPC1 protein.
• Dietary cholesterol uptake is mediated by NPC1L1.
• Understanding cholesterol transport mechanisms is crucial for metabolic health.

Purpose of the Study:

• To investigate the role of inter-domain dynamics in NPC1 and NPC1L1 cholesterol transport.
• To explore how structural constraints affect protein function.
• To identify the binding site of the cholesterol uptake inhibitor ezetimibe.

Main Methods:

• Introduction of single disulfide bonds into NPC1 and NPC1L1.
• Monitoring lysosomal cholesterol efflux using a sensitive assay.
• Analysis of protein structure-function relationships.

Main Results:

• NPC1's N-terminal domain does not need to detach for cholesterol export.
• Constraining extracellular domains or shortening cytoplasmic loops inactivated both NPC1 and NPC1L1.
• Ezetimibe binds to extracellular domains of NPC1L1, blocking cholesterol transport.

Conclusions:

• Cholesterol transport through NPC1/NPC1L1 involves coordinated domain movements.
• Ezetimibe inhibits NPC1L1 by simultaneously engaging multiple domains.
• These findings provide insights into cholesterol homeostasis and drug action.