J. Microbiol. Biotechnol. (2011), 21(10), 1097–1100 doi: 10.4014/jmb.1103.03026 First published online 20 July 2011 In Silico Study of the Ion Channel Formed by Tolaasin I Produced by Pseudomonas tolaasii Jo, Geunhyeong 1 , Doseok Hwang 1 , Sunhee Lee 1 , Yoonkyung Woo 1 , Jiye Hyun 1 , Yeonjoong Yong 1 , Kyungrai Kang 1 , Dong-woon Kim 2 , and Yoongho Lim 1 * Division of Bioscience and Biotechnology, BMIC, Konkuk University, Seoul 143-701, Korea Swine Science Division, National Institute of Animal Science, RDA, Cheonan 330-801, Korea Received: March 15, 2011 / Revised: June 8, 2011 / Accepted: June 16, 2011 A toxin produced by Pseudomonas tolaasii, tolaasin, causes brown blotch disease in mushrooms. Tolaasin forms pores on the cellular membrane and destroys cell structure. Inhibiting the ability of tolaasin to form ion channels may be an effective method to protect against attack by tolaasin. However, it is first necessary to elucidate the three-dimensional structure of the ion channels formed by tolaasin. In this study, the structure of the tolaasin ion channel was determined in silico based on data obtained from nuclear magnetic resonance experiments. Keywords: Tolaasin ion channel, Pseudomonas tolaasii Pseudomonas tolaasii causes brown blotch disease in Agaricus bisporus and Pleurotus ostreatus [1]. A toxin produced by P. tolaasii was identified to be a lipodepsipeptide that forms an ion channel [2]. This toxin was named tolaasin and seven analogs have been identified [1]. Of all the analogs, tolaasin I is the most abundant one. Its primary structure was determined by Nutkins et al. [8], and its three-dimensional structure was determined using nuclear magnetic resonance (NMR) spectroscopy by Jourdan et al. [5]. Tolaasin forms an ion channel that allows ions to penetrate the membrane [4], but it is not known how tolaasin forms these ion channels. To control brown blotch disease caused by P. tolaasii, cultivation houses are typically fumigated and the water is sterilized. However, this treatment cannot completely prevent blotch disease; thus, alternative methods are needed. Because tolaasin causes disease, compounds inhibiting tolaasin have been examined. Several compounds were previously shown to inhibit tolaasin [7] and were primarily glucosylated carboxylic acids. The tolaasin inhibitors bind to tolaasin, which was shown by NMR spectroscopy [7]. It has been known that tolaasin forms pores on cellular membrane and destroys cell structure [3]. Inhibiting the ability of tolaasin to form ion channels may be an effective method to protect against attack by tolaasin. However, it is first necessary to elucidate the three-dimensional structure of ion channels formed by tolaasin. Therefore, in this study, the tolaasin ion channel was built in silico using data obtained from NMR experiments. The three-dimensional (3D) structure of tolaasin I monomer was determined using NMR spectroscopy [5, 7]. Tolaasin I consists of the following amino acid sequence: β-hydroxyoctanoyl-D-But 1 -D-Pro-D-Ser-D-Leu-D-Val-D-Ser- D-Leu-D-Val-L-Val-D-Gln-L-Leu-D-Val-D-But-D-alloThr-L-Ile- L-Hse-D-Dab-L-Lys 18 , where D-But, L-Hse, and D-Dab denote Z-dehydroaminobutyric acid, L-homoserine, and D-2,4- diaminobutyric acid, respectively [4]. This protein contains a lactone ring between D-alloThr 14 and L-Lys 18 , and a left- handed α-helix between D-Pro 2 and D-alloThr 14 . The β- hydroxyoctanoyl chain is parallel to the helix. Since it was first shown that the tolaasin-induced ion channel formation was inhibited by zinc ion, the ability of the zinc ion to penetrate the lactone ring was assessed. The diameter of the backbone of the lactone ring is 5Å × 7Å, but there is no hole in its inner surface owing to the van der Waals clouds. As a result, the ion channel formed by tolaasin I was believed to consist of a trimer or tetramer of tolaasin I. There are a few methods to determine the 3D structure of peptides or proteins. Of these, molecular modeling has both advantages, such as no requirement for real biological samples, and disadvantages, such as low reliability. In this study, we tried to determine the 3D structure of the ion channel formed by tolaasin I using molecular modeling. The structures of the tolaasin I monomer and the ion channel induced by tolaasin I were built using Sybyl (Tripos, St. Louis, MO, USA). Molecular modeling calculations *Corresponding author Phone: +82-2-453-3760; Fax: +82-2-454-3760; E-mail: yoongho@konkuk.ac.kr