Contributions of a Disulfide Bond and a Reduced Cysteine Side Chain to the Intrinsic Activity of the High-Density Lipoprotein Receptor SR-BI

The high-density lipoprotein (HDL) receptor scavenger receptor class B, type I (SR-BI), binds HDL and mediates selective cholesteryl ester uptake. SR-BI’s 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 ≠ 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’s 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’s 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