SUMMARYPathogenesis-related proteins played a pioneering role 50 years ago in the discovery of plant innate immunity as a set of proteins that accumulated upon pathogen challenge. The most abundant of these proteins, PATHOGENESIS-RELATED 1 (PR-1) encodes a small antimicrobial protein that has become, as a marker of plant immune signaling, one of the most referred to plant proteins. The biochemical activity and mode of action of PR-1 proteins has remained elusive, however. Here, we provide genetic and biochemical evidence for the capacity of PR-1 proteins to bind sterols, and demonstrate that the inhibitory effect on pathogen growth is caused by the sequestration of sterol from pathogens. In support of our findings, sterol-auxotroph pathogens such as the oomycete Phytophthora are particularly sensitive to PR-1, whereas sterol-prototroph fungal pathogens become highly sensitive only when sterol biosynthesis is compromised. Our results are in line with previous findings showing that plants with enhanced PR-1 expression are particularly well protected against oomycete pathogens.

including immune defense in mammals and plants, pathogen virulence, sperm maturation and fertilization, venom toxicity, and prostate and brain cancer. Named after the founding members of this protein superfamily (cysteine-rich secretory protein, antigen 5, and pathogenesis-related 1), the CAP superfamily comprises more than 4,500 members in over 1,500 species, and family members are present in all kingdoms of life. Almost all CAP proteins are secreted glycoproteins and they are stable in the extracellular space over a wide range of conditions. All members of this superfamily share a common CAP domain of approximately 150 amino acids, which adopts a unique ␣ -␤ -␣ sandwich fold. The structural conservation of this domain suggests that CAP proteins exert a fundamentally similar function. The molecular mode of action of the CAP proteins, however, has remained enigmatic [for reviews see ( 1, 2 )].CAP proteins share limited sequence identity with each other and are characterized by two PROSITE-recognized sequence motifs, referred to as the CRISP family signature (shaded boxes in Fig. 1 ). 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 fl anked 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 disulfi de bonds. These features are thought to provide the thermal, pH, and proteolytic stability required for the extracellular function of these proteins ( 3, 4 ). The CAP domain harbors four highly 28 February 2014. Published, JLR Papers in Press, March 5, 2014 DOI 10.1194 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 Abbreviations: DOPE, discrete optimized protein energy; GLIPR1, glioma pathogenesis-related protein 1; PR, pathogenesis-related protein; OD, optical density; PDB, protein data base; Pry, pathogen-related yeast; RMSD, root mean square deviation; SC, synthetic complete. Manuscript received 15 January 2014 and in revised form

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.

Members of the CAP superfamily (cysteine-rich secretory proteins, antigen 5, and pathogenesis related 1 proteins), also known as SCP superfamily (sperm-coating proteins), have been implicated in many physiological processes, including immune defenses, venom toxicity, and sperm maturation. Their mode of action, however, remains poorly understood.

The first structure of an S. mansoni venom allergen-like protein is presented.