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

Abstract

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

Plant pathogenesis–related proteins of the cacao fungal pathogen Moniliophthora perniciosa differ in their lipid-binding specificities

“…Our previous studies had revealed that the CBM is important for sterol binding of the yeast Pry1 protein (24). The hallmark of the CBM is the presence of aromatic amino acids in positions 1, 3, and 8 of the motif (øxøxxxxø) (24,28,29).…”

“…Our previous studies had revealed that the CBM is important for sterol binding of the yeast Pry1 protein (24). The hallmark of the CBM is the presence of aromatic amino acids in positions 1, 3, and 8 of the motif (øxøxxxxø) (24,28,29). While the first aromatic residue is conserved in all MpPR-1 proteins, the aromatic residues at positions 3 and 8 of the motif are only conserved in MpPR-1c, MpPR-1d, MpPR-1g, and MpPR-1j, as revealed by the sequence alignment shown in Fig.…”

“…M. perniciosa PR-1 proteins harbor a fold that is characteristic of all CAP proteins, an alphabeta-alpha sandwich fold (26,33). The sterolbinding domain within the CAP domain is composed of a flexible loop containing aromatic amino acids (24). This caveolinbinding motif is conserved in the MpPR-1 members that binds sterols, but in members that do not bind cholesterol, the CBM contains substitutions of the important residues at position 3 and 8 of the motif.…”

“…The sterol and fatty acid binding and export function of the yeast CAP proteins is confined to the CAP domain, because expression of the CAP domain alone is sufficient to rescue the sterol and fatty acid export phenotype of a pry1 pry2 double mutant, and the CAP domain of Pry1 alone binds sterols and fatty acids at distinct sites in vitro (19,22,23). Sterol binding by Pry proteins requires the displacement of a flexible loop containing aromatic amino acids, termed the caveolin-binding motif (CBM) within the CAP domain, since point mutations within this motif abolish sterol export and binding, whereas fatty acid binding takes place in the groove between two parallel running helices, 1 and 3 (19,24). Taken together, these data indicate that CAP proteins may exert an immunomodulatory function through binding hydrophobic ligands, such as sterols or related small hydrophobic compounds and fatty acids and their derivatives such as leukotrienes (19,23,25).…”

Cholesterol-Binding by the Yeast CAP Family Member Pry1 Requires the Presence of an Aliphatic Side Chain on Cholesterol

“…Point mutations within this motif abrogate sterol export and binding while mutations of residues located outside the CBM had no effect on lipid export and binding. The CBM thus appears to play a key role in the ability of CAP proteins to bind cholesterol [5][6][7].…”