Extracellular loop C of NPC1L1 is important for binding to ezetimibe

Proc Natl Acad Sci U S A. 2008 Aug 12;105(32):11140-5. doi: 10.1073/pnas.0800936105. Epub 2008 Aug 5.


Niemann-Pick C1-like protein (NPC1L1) mediates the absorption of dietary cholesterol in the proximal region of the intestine, a process that is blocked by cholesterol absorption inhibitors (CAIs), including ezetimibe (EZE). Using a proteomic approach, we demonstrate that NPC1L1 is the protein to which EZE and its analogs bind. Next, we determined the site of interaction of EZE analogs with NPC1L1 by exploiting the different binding affinities of mouse and dog NPC1L1 for the radioligand analog of EZE, [(3)H]AS. Chimeric and mutational studies indicate that high-affinity binding of [(3)H]AS to dog NPC1L1 depends on molecular determinants present in a 61-aa region of a large extracellular domain (loop C), where Phe-532 and Met-543 appear to be key contributors. These data suggest that the [(3)H]AS-binding site resides in the intestinal lumen and are consistent with preclinical data demonstrating in vivo efficacy of a minimally bioavailable CAI. Furthermore, these determinants of [(3)H]AS binding lie immediately adjacent to a hotspot of human NPC1L1 polymorphisms correlated with hypoabsorption of cholesterol. These observations, taken together with the recently described binding of cholesterol to the N terminus (loop A) of the close NPC1L1 homologue, NPC1, may provide a molecular basis for understanding EZE inhibition of NPC1L1-mediated cholesterol absorption. Specifically, EZE binding to an extracellular site distinct from where cholesterol binds prevents conformational changes in NPC1L1 that are necessary for the translocation of cholesterol across the membrane.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • Anticholesteremic Agents / pharmacology*
  • Azetidines / pharmacology*
  • Binding Sites / drug effects
  • Binding Sites / genetics
  • Biological Transport, Active / drug effects
  • Biological Transport, Active / genetics
  • Cell Line
  • Cell Membrane / genetics
  • Cell Membrane / metabolism
  • Cholesterol, Dietary / metabolism*
  • Dogs
  • Ezetimibe
  • Intestinal Absorption / drug effects
  • Intestinal Absorption / genetics
  • Membrane Proteins / antagonists & inhibitors*
  • Membrane Proteins / metabolism*
  • Membrane Transport Proteins / genetics
  • Membrane Transport Proteins / metabolism*
  • Mice
  • Mutation
  • Polymorphism, Genetic
  • Protein Binding / drug effects
  • Protein Binding / genetics
  • Protein Structure, Secondary / genetics
  • Protein Structure, Tertiary / genetics
  • Proteomics / methods


  • Anticholesteremic Agents
  • Azetidines
  • Cholesterol, Dietary
  • Membrane Proteins
  • Membrane Transport Proteins
  • NPC1L1 protein, human
  • Npc1l1 protein, mouse
  • Ezetimibe