The caveolin-binding motif of the pathogen-related yeast protein Pry1, a member of the CAP protein superfamily, is required for in vivo export of cholesteryl acetate

Vineet Choudhary,Rabih Darwiche,David Gfeller,Vincent Zoete,Olivier Michielin,Roger Schneiter

doi:10.1194/jlr.m047126

Vineet Choudhary, Rabih Darwiche + Show 4 more

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https://doi.org/10.1194/jlr.m047126

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Abstract

Proteins belonging to the CAP superfamily are present in all kingdoms of life and have been implicated in different physiological processes. Their molecular mode of action, however, is poorly understood. Saccharomyces cerevisiae expresses three members of this superfamily, pathogen-related yeast (Pry)1, -2, and -3. We have recently shown that Pry function is required for the secretion of cholesteryl acetate and that Pry proteins bind cholesterol and cholesteryl acetate, suggesting that CAP superfamily members may generally act to bind sterols or related small hydrophobic compounds. Here, we analyzed the mode of sterol binding by Pry1. Computational modeling indicates that ligand binding could occur through displacement of a relatively poorly conserved flexible loop, which in some CAP family members displays homology to the caveolin-binding motif. Point mutations within this motif abrogated export of cholesteryl acetate but did not affect binding of cholesterol. Mutations of residues located outside the caveolin-binding motif, or mutations in highly conserved putative catalytic residues had no effect on export of cholesteryl acetate or on lipid binding. These results indicate that the caveolin-binding motif of Pry1, and possibly of other CAP family members, is crucial for selective lipid binding and that lipid binding may occur through displacement of the loop containing this motif.

Highlights

Proteins belonging to the CAP superfamily are present in all kingdoms of life and have been implicated in different physiological processes
As a first step toward an understanding how the yeast CAP family member, Pry1, can bind a hydrophobic ligand such as cholesterol, we modeled the structure of the CAP
Analysis of the model of the yeast protein, did not reveal a potential hydrophobic pocket that could accommodate cholesterol, and all reported hydrophobic patches are buried inside the structure and not directly surface accessible, suggesting that cholesterol does not bind by sticking to a hydrophobic patch at the protein surface, but that binding of the lipid is likely accompanied by significant structural rearrangement of the protein, as has been observed in other cholesterol binding proteins [e.g., ␤-elicitin cryptogein [17]]

Summary

Introduction

Proteins belonging to the CAP superfamily are present in all kingdoms of life and have been implicated in different physiological processes. Mutations of residues located outside the caveolin-binding motif, or mutations in highly conserved putative catalytic residues had no effect on export of cholesteryl acetate or on lipid binding These results indicate that the caveolin-binding motif of Pry, and possibly of other CAP family members, is crucial for selective lipid binding and that lipid binding may occur through displacement of the loop containing this motif.—Choudhary, V., R. An early comparison of the structure of the plant pathogenesis-related protein (PR)-1 with that of the human glioma pathogenesis-related protein 1 (GLIPR1) revealed that the small and structurally conserved 17–21 kDa CAP domain adopts a unique ␣-␤-␣ sandwich fold, in which a three-stranded anti-parallel ␤-sheet is flanked by three helices on one side, and a fourth helix on the other [3] This three-stacked layer is stabilized by hydrophobic interactions, multiple hydrogen bonds, and by two highly conserved disulfide bonds.

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