Contributions of a Disulfide Bond and a Reduced Cysteine Side Chain to the Intrinsic Activity of the High-Density Lipoprotein Receptor SR-BI
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- 作者：Miao Yu ; Thomas Y. Lau ; Steven A. Carr ; Monty Krieger
- 刊名：Biochemistry
- 出版年：2012
- 出版时间：December 18, 2012
- 年：2012
- 卷：51
- 期：50
- 页码：10044-10055
- 全文大小：442K
- 年卷期：v.51,no.50(December 18, 2012)
- ISSN：1520-4995
文摘
The high-density lipoprotein (HDL) receptor scavenger receptor class B, type I (SR-BI), binds HDL and mediates selective cholesteryl ester uptake. SR-BI鈥檚 structure and mechanism are poorly understood. We used mass spectrometry to assign the two disulfide bonds in SR-BI that connect cysteines within the conserved Cys321-Pro322-Cys323 (CPC) motif and connect Cys280 to Cys334. We used site-specific mutagenesis to evaluate the contributions of the CPC motif and the side chain of extracellular Cys384 to HDL binding and lipid uptake. The effects of CPC mutations on activity were context-dependent. Full wild-type (WT) activity required Pro322 and Cys323 only when Cys321 was present. Reduced intrinsic activities were observed for CXC and CPX, but not XXC, XPX, or XXX mutants (X 鈮?WT residue). Apparently, a free thiol side chain at position 321 that cannot form an intra-CPC disulfide bond with Cys323 is deleterious, perhaps because of aberrant disulfide bond formation. Pro322 may stabilize an otherwise strained CPC disulfide bond, thus supporting WT activity, but this disulfide bond is not absolutely required for normal activity. C384X (X = S, T, L, Y, G, or A) mutants exhibited altered activities that varied with the side chain鈥檚 size: larger side chains phenocopied WT SR-BI treated with its thiosemicarbazone inhibitor BLT-1 (enhanced binding, weakened uptake); smaller side chains produced almost inverse effects (increased uptake:binding ratio). C384X mutants were BLT-1-resistant, supporting the proposal that Cys384鈥檚 thiol interacts with BLT-1. We discuss the implications of our findings on the functions of the extracellular loop cysteines in SR-BI and compare our results to those presented by other laboratories.