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The N-terminal domain of NPC1L1 protein binds cholesterol and plays essential roles in cholesterol uptake.

ABSTRACT: Niemann-Pick C1-like 1 (NPC1L1) is a multitransmembrane protein playing a crucial role in dietary and biliary cholesterol absorption. Cholesterol promotes the formation and endocytosis of NPC1L1-flotillin-cholesterol membrane microdomains, which is an early step in cholesterol uptake. How cholesterol is sensed in this step is unknown. Here, we find that the N-terminal domain (NTD) of NPC1L1 binds cholesterol. Mutation of residue Leu-216 in NPC1L1-NTD eliminates cholesterol binding, decreases the formation of NPC1L1-flotillin-cholesterol membrane microdomains, and prevents NPC1L1-mediated cholesterol uptake in culture cells and mice livers. NPC1L1-NTD specifically binds cholesterol but not plant sterols, which may account for the selective cholesterol absorption in intestine. Furthermore, 25- or 27-hydroxycholesterol competes with cholesterol to bind NPC1L1-NTD and inhibits the cholesterol induced endocytosis of NPC1L1. Together, these results demonstrate that plasma membrane-localized NPC1L1 binds exogenous cholesterol via its NTD, and facilitates the formation of NPC1L1-flotillin-cholesterol membrane microdomains that are then internalized into cells through the clathrin-AP2 pathway. Our study uncovers the mechanism of cholesterol sensing by NPC1L1 and proposes a mechanism for selective cholesterol absorption.

SUBMITTER: Zhang JH

PROVIDER: S-EPMC3137082 | biostudies-literature | 2011 Jul

REPOSITORIES: biostudies-literature

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The N-terminal domain of NPC1L1 protein binds cholesterol and plays essential roles in cholesterol uptake.

Zhang Jin-Hui JH Ge Liang L Qi Wei W Zhang Liqing L Miao Hong-Hua HH Li Bo-Liang BL Yang Maojun M Song Bao-Liang BL

The Journal of biological chemistry 20110520 28

Niemann-Pick C1-like 1 (NPC1L1) is a multitransmembrane protein playing a crucial role in dietary and biliary cholesterol absorption. Cholesterol promotes the formation and endocytosis of NPC1L1-flotillin-cholesterol membrane microdomains, which is an early step in cholesterol uptake. How cholesterol is sensed in this step is unknown. Here, we find that the N-terminal domain (NTD) of NPC1L1 binds cholesterol. Mutation of residue Leu-216 in NPC1L1-NTD eliminates cholesterol binding, decreases the ...[more]

PMID: 21602275

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Project description:Cholesterol is a fat-like substance with a pivotal physiological relevance in humans, and its homeostasis is tightly regulated by various cellular processes, including the import in the small intestine and the reabsorption in the biliary ducts by the Niemann-Pick C1 Like 1 (NPC1L1) importer. NPC1L1 can mediate the absorption of a variety of sterols but strikingly exhibits a large sensitivity to cholesterol epimerization. This study examines the molecular basis of the epimerization-related selective binding of cholesterol by combining extended unbiased molecular dynamics simulations of the apo and holo species of the N-terminal domain of wild-type NPC1L1, in conjunction with relative binding free energy, umbrella sampling, and well-tempered metadynamics calculations. The analysis of the results discloses the existence of two distinct binding modes for cholesterol and epi-cholesterol. The former binds deeper in the cavity, forming key hydrogen-bond interactions with Q95, S56, and a water molecule. In contrast, epi-cholesterol is shifted ca. 3 Å to the mouth of the cavity and the transition to the Q95 site is prevented by an energetic barrier of 4.1 kcal·mol-1. Thus, the configuration of the hydroxyl group of cholesterol, together with the presence of a structural water molecule, is a key feature for effective absorption. Finally, whereas these findings may seemingly be challenged by single-point mutations that impair cholesterol transport but have a mild impact on the binding of cholesterol to the Q95 binding site, our results reveal that they have a drastic influence on the conformational landscape of the α8/β7 loop in the apo species compared to the wild-type protein. Overall, the results give support to the functional role played by the α8/β7 loop in regulating the access of ligands to NPC1L1, and hence to interpreting the impact of these mutations on diseases related to disruption of sterol absorption, paving the way to understanding certain physiological dysfunctions.

Project description:BackgroundThe non-receptor tyrosine kinase Abelson (Abl) is a key player in oncogenesis, with kinase inhibitors serving as paradigms of targeted therapy. Abl also is a critical regulator of normal development, playing conserved roles in regulating cell behavior, brain development and morphogenesis. Drosophila offers a superb model for studying Abl's normal function, because, unlike mammals, there is only a single fly Abl family member. In exploring the mechanism of action of multi-domain scaffolding proteins like Abl, one route is to define the roles of their individual domains. Research into Abl's diverse roles in embryonic morphogenesis revealed many surprises. For instance, kinase activity, while important, is not crucial for all Abl activities, and the C-terminal F-actin binding domain plays a very modest role. This turned our attention to one of Abl's least understood features-the long intrinsically-disordered region (IDR) linking Abl's kinase and F-actin binding domains. The past decade revealed unexpected, important roles for IDRs in diverse cell functions, as sites of posttranslational modifications, mediating multivalent interactions and enabling assembly of biomolecular condensates via phase separation. Previous work deleting conserved regions in Abl's IDR revealed an important role for a PXXP motif, but did not identify any other essential regions.MethodsHere we extend this analysis by deleting the entire IDR, and asking whether Abl∆IDR rescues the diverse roles of Abl in viability and embryonic morphogenesis in Drosophila.ResultsThis revealed that the IDR is essential for embryonic and adult viability, and for cell shape changes and cytoskeletal regulation during embryonic morphogenesis, and, most surprisingly, revealed a role in modulating protein stability.ConclusionOur data provide new insights into the role of the IDR in an important signaling protein, the non-receptor kinase Abl, suggesting that it is essential for all aspects of protein function during embryogenesis, and revealing a role in protein stability. These data will stimulate new explorations of the mechanisms by which the IDR regulates Abl stability and function, both in Drosophila and also in mammals. They also will stimulate further interest in the broader roles IDRs play in diverse signaling proteins. Video Abstract.

Dataset Information

The N-terminal domain of NPC1L1 protein binds cholesterol and plays essential roles in cholesterol uptake.

Publications

The N-terminal domain of NPC1L1 protein binds cholesterol and plays essential roles in cholesterol uptake.

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