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Proc Natl Acad Sci U S A DOI:10.1073/pnas.1109078108

Exoplasmic cysteine Cys384 of the HDL receptor SR-BI is critical for its sensitivity to a small-molecule inhibitor and normal lipid transport activity.

Publication TypeJournal Article
Year of Publication2011
AuthorsYu, M, Romer, KA, Nieland, TJF, Xu, S, Saenz-Vash, V, Penman, M, Yesilaltay, A, Carr, SA, Krieger, M
JournalProc Natl Acad Sci U S A
Date Published2011 Jul 26
KeywordsAmino Acid Substitution, Animals, Binding Sites, Biological Transport, Active, Cercopithecus aethiops, COS Cells, Cyclopentanes, Cysteine, Humans, In Vitro Techniques, Lipid Metabolism, Lipoproteins, HDL, Mass Spectrometry, Mice, Models, Molecular, Mutagenesis, Site-Directed, Mutant Proteins, Recombinant Proteins, Scavenger Receptors, Class B, Thiosemicarbazones

The HDL receptor, scavenger receptor, class B, type I (SR-BI), is a homooligomeric cell surface glycoprotein that controls HDL structure and metabolism by mediating the cellular selective uptake of lipids, mainly cholesteryl esters, from HDL. The mechanism underlying SR-BI-mediated lipid transfer, which differs from classic receptor-mediated endocytosis, involves a two-step process (binding followed by lipid transport) that is poorly understood. Our previous structure/activity analysis of the small-molecule inhibitor blocker of lipid transport 1 (BLT-1), which potently (IC(50) ∼ 50 nM) blocks SR-BI-mediated lipid transport, established that the sulfur in BLT-1's thiosemicarbazone moiety was essential for activity. Here we show that BLT-1 is an irreversible inhibitor of SR-BI, raising the possibility that cysteine(s) in SR-BI interact with BLT-1. Mass spectrometric analysis of purified SR-BI showed two of its six exoplasmic cysteines have free thiol groups (Cys251 and Cys384). Converting Cys384 (but not Cys251) to serine resulted in complete BLT-1 insensitivity, establishing that the unique molecular target of BLT-1 inhibition of cellular SR-BI dependent lipid transport is SR-BI itself. The C384S substitution reduced the receptor's intrinsic lipid uptake activity by approximately 60% without dramatically altering its surface expression, homooligomerization, or HDL binding. Thus, a small-molecule screening approach identified a key residue in SR-BI involved in lipid transport, providing a powerful springboard into the analyses of the structure and mechanism of SR-BI, and highlighting the power of this approach for such analyses.


Alternate JournalProc. Natl. Acad. Sci. U.S.A.
PubMed ID21746906
PubMed Central IDPMC3145699