Structural and functional characterization of the CAP domain of pathogen-related yeast 1 (Pry1) protein.

Rabih Darwiche,Elissa M Hudspeth,Oluwatoyin A Asojo,Alan Kelleher,Roger Schneiter

doi:10.1038/srep28838

Rabih Darwiche, Elissa M Hudspeth + Show 3 more

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https://doi.org/10.1038/srep28838

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Abstract

The production, crystal structure, and functional characterization of the C-terminal cysteine-rich secretory protein/antigen 5/pathogenesis related-1 (CAP) domain of pathogen-related yeast protein-1 (Pry1) from Saccharomyces cerevisiae is presented. The CAP domain of Pry1 (Pry1CAP) is functional in vivo as its expression restores cholesterol export to yeast mutants lacking endogenous Pry1 and Pry2. Recombinant Pry1CAP forms dimers in solution, is sufficient for in vitro cholesterol binding, and has comparable binding properties as full-length Pry1. Two crystal structures of Pry1CAP are reported, one with Mg2+ coordinated to the conserved CAP tetrad (His208, Glu215, Glu233 and His250) in spacegroup I41 and the other without divalent cations in spacegroup P6122. The latter structure contains four 1,4-dioxane molecules from the crystallization solution, one of which sits in the cholesterol binding site. Both structures reveal that the divalent cation and cholesterol binding sites are connected upon dimerization, providing a structural basis for the observed Mg2+-dependent sterol binding by Pry1.

Highlights

IntroductionMembers of the eukaryotic CAP (cysteine-rich secretory protein/antigen 5/pathogenesis related-1) or SCP/TAPS (Sperm-coating protein/Tpx/antigen 5/pathogenesis related-1/Sc7) superfamily have a wide range of physiological activities including sperm maturation, fertilization, fungal virulence, cellular defense, and immune evasion[1,2,3,4,5,6]
Members of the eukaryotic cysteine-rich secretory protein/antigen 5/pathogenesis related-1 (CAP) or SCP/TAPS (Sperm-coating protein/Tpx/antigen 5/pathogenesis related-1/Sc7) superfamily have a wide range of physiological activities including sperm maturation, fertilization, fungal virulence, cellular defense, and immune evasion[1,2,3,4,5,6]
CAP1 and CAP2 are defined in the PROSITE database as CRISP motifs, while CAP3 and CAP4 are additional CAP motifs defined by Gibbs and colleagues[8]

Summary

Introduction

Members of the eukaryotic CAP (cysteine-rich secretory protein/antigen 5/pathogenesis related-1) or SCP/TAPS (Sperm-coating protein/Tpx/antigen 5/pathogenesis related-1/Sc7) superfamily have a wide range of physiological activities including sperm maturation, fertilization, fungal virulence, cellular defense, and immune evasion[1,2,3,4,5,6]. While point mutations within the CBM abrogated sterol binding and export, mutations of residues located outside the CBM including highly conserved putative catalytic residues have minimal effect on lipid binding and sterol export[23]. These studies defined the CBM as a crucial motif for lipid binding in vitro and sterol export in vivo[22,23]. As part of our ongoing efforts to characterize the lipid binding properties of this ubiquitous superfamily of proteins[6,11,13,25,26,27,28], we present the structural and functional characterization of the CAP domain of Pry[1] (Pry1CAP) from Saccharomyces cerevisiae

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