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

Saha, P.; Shumate, J. L.; Caldwell, J. G.; Elghobashi-Meinhardt, N.; Lu, A.; Zhang, L.; Olsson, N.; Elias, J. E.; Pfeffer, S. R.

doi:10.1101/2020.03.26.009217

bioRxiv · 10.1101/2020.03.26.009217

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

Saha, P.·Shumate, J. L.·Caldwell, J. G.·Elghobashi-Meinhardt, N.·Lu, A.·Zhang, L.·Olsson, N.·Elias, J. E.·Pfeffer, S. R.

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Abstract

Transport of LDL-derived cholesterol from lysosomes into the cytoplasm requires NPC1 protein; NPC1L1 mediates uptake of dietary cholesterol. We introduced single disulfide bonds into NPC1 and NPC1L1 to explore the importance of inter-domain dynamics in cholesterol transport. Using a sensitive method to monitor lysosomal cholesterol efflux, we find that NPC1s N-terminal domain need not release from the rest of the protein for efficient cholesterol export. Either introducing single disulfide bonds to constrain lumenal/extracellular domains or shortening a cytoplasmic loop abolishes transport activity by both NPC1 and NPC1L1. The widely prescribed cholesterol uptake inhibitor, Ezetimibe, blocks NPC1L1; we show that interface residues that lie at the interface between NPC1L1s three extracellular domains comprise the drugs binding site. These data support a model in which cholesterol passes through the cores of NPC1/NPC1L1 proteins; concerted movement of various domains is needed for transfer and Ezetimibe blocks transport by binding to multiple domains simultaneously.

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Saha, P., Shumate, J. L., Caldwell, J. G., Elghobashi-Meinhardt, N., Lu, A., Zhang, L., Olsson, N., Elias, J. E., Pfeffer, S. R.. 2020-03-29. Inter-domain dynamics drive cholesterol transport by NPC1 and NPC1L1 proteins. https://doi.org/10.1101/2020.03.26.009217

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