Binding of Fatty Acids to -Cryptogein: Quantitative Structure-Activity Relationships and Design of Selective Protein Mutants Petr Dobes ˇ, †,‡ Jan Kmunı ´c ˇek, † Vladimı ´r Mikes ˇ, ‡ andJirˇı ´ Damborsky ´* ,† National Centre for Biomolecular Research and Department of Biochemistry, Faculty of Science, Masaryk University, Kotlarska 2, 611 37 Brno, Czech Republic Received May 22, 2004 Binding of fatty acids to cryptogein, the proteinaceous elicitor from Phytophthora, was studied by using molecular docking and quantitative structure-activity relationships analysis. Fatty acids bind to the groove located inside the cavity of cryptogein. The structure-activity model was constructed for the set of 27 different saturated and unsaturated fatty acids explaining 87% (81% cross-validated) of the quantitative variance in their binding affinity. The difference in binding between saturated and unsaturated fatty acids was described in the model by three electronic descriptors: the energy of the lowest unoccupied molecular orbital, the energy of the highest occupied molecular orbital, and the heat of formation. The presence of double bonds in the ligand generally resulted in stronger binding. The difference in binding within the group of saturated fatty acids was explained by two steric descriptors, i.e., ellipsoidal volume and inertia moment of length, and one hydrophobicity descriptor, i.e., lipophility. The developed model predicted strong binding for two biologically important molecules, geranylgeranyol and farnesol playing an important role in plant signaling as lipid anchors of some membrane proteins. Elicitin mutants selectively binding only one type of ligand were designed for future experimental studies. INTRODUCTION Study of relationships between a pathogen and a plant is important for protection of plants against infections. Elicitors are the molecules participating in the plant-pathogen interac- tions. Secretion of elicitors by a pathogen to the surrounding environment and their interaction with plant cells can induce plant hypersensitive reaction. Elicitins make up the highly conserved family of protein elicitors. 1 They are secreted specifically by the fungi Oomycete genera Pythium and Phytophthora. 2,3 The biological function of elicitins is currently unknown. The study of physiological effects on tobacco plants revealed that elicitins have the ability to induce so-called “systemic acquired resistance” against the pathogen attack accompanied by restricted leaf necrosis. 3,4 The re- sponse is induced by the interaction of elicitins with a putative receptor located on the cytoplasmic membrane of tobacco cells 5 composed of a calcium channel 6 and a glycoprotein. 7 The transfer of a signal through the receptor triggers phosphorylation-dephosphorylation cascades in the tobacco resulting in alkalinization of the extracellular medium, efflux of potassium and chloride ions, influx of calcium, production of the active species from oxygen, 5,8 and changes in the composition of the cell wall. 9 The primary structure of mature elicitins is composed of 98 amino acids (10 kDa) that are interconnected by three disulfide bridges. Elicitins can be classified according to their pI as R-elicitins (pI < 7) and -elicitins (pI > 7). The -elicitins generally induce a greater necrotic effect than the R-elicitins 10 due to the presence of polar amino acids at necrotic sites located on the protein surface. 11,12 The three-dimensional structures of two elicitin family members, cryptogein and cinnamomin, were determined by X-ray crystallography. 13,14 The structure of cryptogein was also studied by NMR spectroscopy. 15 The structures of elicitins are composed of five R-helices and one -sheet arranged in a unique protein fold. A hydrophobic cavity is located in the protein core and connected with the protein surface by a tunnel. The original proposal that elicitins may facilitate transfer of sterols 16 was corroborated by the crystal structures of cryptogein in complex with dehydroergosterol (DHE) 17 and cholesterol. 18 Binding of ligands to the cavity seems to be essential for consecutive association of the elicitin with a receptor and induction of a biological response in a plant. 19 Besides sterols, also fatty acids (FAs) can bind to the internal cavity of elicitins 20 making them functionally similar to the family of plant lipid transfer proteins. Interest- ingly, plant lipid transfer proteins can associate with the same receptor in tobacco as elicitins, 21 and they can bind FAs and phospholipids but not sterols. Here we study binding of FAs to the protein cryptogein at atomic detail. Models of the FA-crytogein complexes were constructed by molecular docking to obtain information about position and conformation of ligands inside the cavity. These models consequently served for design of mutant proteins binding selectively one type of ligand. The physicochemical properties important for the binding were identified by quantitative structure-activity relationships (QSAR) analysis, and the model served for prediction of binding affinities of two lipids, farnesol and geranylgeranyol, that play an important role in plant signaling. 22 * Corresponding author e-mail: jiri@chemi.muni.cz. † National Centre for Biomolecular Research. ‡ Department of Biochemistry. 2126 J. Chem. Inf. Comput. Sci. 2004, 44, 2126-2132 10.1021/ci049832x CCC: $27.50 © 2004 American Chemical Society Published on Web 10/07/2004