Effect of pH on the Pore Forming Activity and Conformational Stability of Ostreolysin, a Lipid Raft-Binding Protein from the Edible Mushroom Pleurotus ostreatus ²,‡ Sabina Berne, § Kristina Sepc ˇic ´, § Gregor Anderluh, § Tom Turk, § Peter Mac ˇek, § and Natas ˇa Poklar Ulrih* ,| Department of Biology, Biotechnical Faculty, UniVersity of Ljubljana, Vee ` na pot 111, 1000 Ljubljana, SloVenia, and Department of Food Science and Technology, Biotechnical Faculty, UniVersity of Ljubljana, JamnikarjeVa 101, 1000 Ljubljana, SloVenia ReceiVed May 30, 2005; ReVised Manuscript ReceiVed June 28, 2005 ABSTRACT: Ostreolysin, a pore-forming protein from the edible oyster mushroom (Pleurotus ostreatus), is a member of the aegerolysin protein family, a novel group of small acidic proteins found in bacteria, molds, mushrooms, and plants. It binds to lipid rafts and interacts specifically with cholesterol-rich lipid domains. In this study, ostreolysin was classified as a single-domain all--structured protein on the basis of cDNA sequencing. pH-induced and thermally induced unfolding of ostreolysin was studied by means of CD, UV absorption, and intrinsic tryptophan fluorescence to characterize conformational transitions associated with its functional properties, i.e., binding to lipid membranes, pore forming activity on lipid vesicles, and hemolysis. At 25 °C and between pH 6 and 9, ostreolysin adopted a monomeric and thermodynamically stable nativelike conformation, characterized by rigid tertiary structure and predomi- nantly -sheet secondary structure. Between pH 2 and 3, the protein underwent an irreversible transition to a partially unfolded, molten globule-like state which bound ANS, and exhibited disrupted tertiary structure and enhanced non-native R-helical structure. Functional studies showed that, unlike colicins and some other bacterial pore-forming toxins, the acid-induced molten globule-like state of ostreolysin is not relevant for lipid binding and pore formation. Instead, the compact native state was necessary for binding to cholesterol/sphingomyelin multilamellar vesicles, optimally in the pH range from 6 to 7, and for pore formation and hemolysis, maximally between pH 7 and 8. Characterization of ostreolysin and aegerolysin (1, 2), ∼15 kDa acidic proteins from the edible mushrooms Pleurotus ostreatus (oyster mushroom) and Agrocybe aegerita (black poplar mushroom), respectively, led to them being assigned to a novel aegerolysin protein family (PF06355 and IPR 009413). Other family members are Asp-hemolysin from the pathogenic mould Aspergillus fumigatus (3), pleurotolysin A (PlyA) from P. ostreatus, an ostreolysin isoform (4, 5), two Clostridium bifermentans hemolysin-like proteins (6), a hypothetical protein PA0122 from Pseudomonas aerugi- nosa (7), a putative protein from Neurospora crassa (8), and proteins predicted from the expressed sequence tag (EST) 1 and genomic nucleotide sequences (Figure 1). Aegerolysin and ostreolysin are expressed specifically during the forma- tion of primordia and fruiting bodies (1, 2). Immunolocal- ization showed that they are concentrated in the growing regions of the basidiocarp, especially in basidia and spores (9). It has been proposed that aegerolysin is involved in the compaction of hyphae during the formation of fruiting body primordia (2), and that C. bifermentans hemolysin-like proteins are related to bacterial sporulation (6). However, the exact biological function of aegerolysins remains to be resolved. Interaction with lipids and pore forming activity are characteristic of certain aegerolysins. Asp-hemolysin was suggested to bind specifically to lysophosphatidylcholine, a component of oxidized low-density lipoprotein (3, 10, 11). Aegerolysin, ostreolysin, and PlyA were reported to lyse erythrocytes and several transformed cell lines by creating transmembrane pores (1, 4, 12). Binding and pore forming activity of ostreolysin, inhibited by the presence of unsatur- ated glycerophosphatides and lysophospholipids (12, 13), were found to be specific to liquid-ordered microdomains, structures emerging in cholesterol- and ergosterol-rich lipid membranes, and lipid rafts (13). PlyA has been suggested to be a sphingomyelin-specific cytolysin, acting as a bicom- ponent cytolysin in concert with 59 kDa pleurotolysin B (PlyB). On erythrocyte membranes, PlyA and PlyB (when ² This work was supported by the Ministry of Higher Education, Science and Technology of the Republic of Slovenia. ‡ GenBank entry AY818435 (P. ostreatus ostreolysin mRNA, partial coordinates). * To whom correspondence should be addressed. Telephone: +386 1 423 11 61. Fax: +386 1 256 62 98. E-mail: natasa.poklar@ bf.uni-lj.si. § Department of Biology. | Department of Food Science and Technology. 1 Abbreviations: ANS, 8-anilinonaphthalene-1-sulfonic acid; CD, circular dichroism; Chol, cholesterol; DTT, dithiothreitol; EST, ex- pressed sequence tag; GnHCl, guanidine hydrochloride; ∆C p°, apparent standard heat capacity difference between the denatured and native state of ostreolysin; ∆G° d(T), Gibbs free energy; ∆H°d(T), denaturation enthalpy; ∆S°d(T), denaturation entropy; ∆H°vH, van’t Hoff enthalpy of denaturation; HEPES, 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid, monosodium salt; LUV, large unilamellar vesicles; MES, 2-mor- pholinoethanesulfonic acid; MLV, multilamellar vesicles; MW, mo- lecular weight; SEL, sheep erythrocyte lipids; SM, sphingomyelin; T d, temperature at the denaturation midpoint. 11137 Biochemistry 2005, 44, 11137-11147 10.1021/bi051013y CCC: $30.25 © 2005 American Chemical Society Published on Web 07/30/2005