title: Crystal structure of an antifungal osmotin-like protein from Calotropis procera and its effects on Fusarium solani spores, as revealed by atomic force microscopy: Insights into the mechanism of action.
abstract: CpOsm is an antifungal osmotin/thaumatin-like protein purified from the latex of Calotropis procera. The protein is relatively thermostable and retains its antifungal activity over a wide pH range; therefore, it may be useful in the development of new antifungal drugs or transgenic crops with enhanced resistance to phytopathogenic fungi. To gain further insight into the mechanism of action of CpOsm, its three-dimensional structure was determined, and the effects of the protein on Fusarium solani spores were investigated by atomic force microscopy (AFM). The atomic structure of CpOsm was solved at a resolution of 1.61Å, and it contained 205 amino acid residues and 192 water molecules, with a final R-factor of 18.12% and an Rfree of 21.59%. The CpOsm structure belongs to the thaumatin superfamily fold and is characterized by three domains stabilized by eight disulfide bonds and a prominent charged cleft, which runs the length of the front side of the molecule. Similarly to other antifungal thaumatin-like proteins, the cleft of CpOsm is predominantly acidic. AFM images of F. solani spores treated with CpOsm resulted in striking morphological changes being induced by the protein. Spores treated with CpOsm were wrinkled, and the volume of these cells was reduced by approximately 80%. Treated cells were covered by a shell of CpOsm molecules, and the leakage of cytoplasmic content from these cells was also observed. Based on the structural features of CpOsm and the effects that the protein produces on F. solani spores, a possible mechanism of action is suggested and discussed.
authors.affiliation: Departamento de Bioquímica e Biologia Molecular, Centro de Ciências, Universidade Federal do Ceará, Fortaleza, Ceará, Brazil.
authors.forename: Marcio V
authors.initials: MV
authors.lastname: Ramos
authors.affiliation: Departamento de Bioquímica e Biologia Molecular, Centro de Ciências, Universidade Federal do Ceará, Fortaleza, Ceará, Brazil.
authors.forename: Raquel S B
authors.initials: RS
authors.lastname: de Oliveira
authors.affiliation: Instituto de Física de São Carlos, Universidade de São Paulo, 13563-120 São Carlos, São Paulo, Brazil.
authors.forename: Humberto M
authors.initials: HM
authors.lastname: Pereira
authors.affiliation: Núcleo de Biologia Experimental, Universidade de Fortaleza, Fortaleza, Ceará, Brazil.
authors.forename: Frederico B M B
authors.initials: FB
authors.lastname: Moreno
authors.affiliation: Núcleo de Biologia Experimental, Universidade de Fortaleza, Fortaleza, Ceará, Brazil.
authors.forename: Marina D P
authors.initials: MD
authors.lastname: Lobo
authors.affiliation: Departamento de Física, Centro de Ciências, Universidade Federal do Ceará, Caixa Postal 6030, Campus do Pici, 60440-900 Fortaleza, Ceará, Brazil.
authors.forename: Luciana M
authors.initials: LM
authors.lastname: Rebelo
authors.affiliation: Diamond Light Source, Harwell Science and Innovation Campus Didcot, Oxfordshire OX11 0DE, United Kingdom.
authors.forename: José
authors.initials: J
authors.lastname: Brandão-Neto
authors.affiliation: Departamento de Física, Centro de Ciências, Universidade Federal do Ceará, Caixa Postal 6030, Campus do Pici, 60440-900 Fortaleza, Ceará, Brazil.
authors.forename: Jeanlex S
authors.initials: JS
authors.lastname: de Sousa
authors.affiliation: Núcleo de Biologia Experimental, Universidade de Fortaleza, Fortaleza, Ceará, Brazil.
authors.forename: Ana C O
authors.initials: AC
authors.lastname: Monteiro-Moreira
authors.affiliation: Departamento de Bioquímica e Biologia Molecular, Centro de Ciências, Universidade Federal do Ceará, Fortaleza, Ceará, Brazil.
authors.forename: Cléverson D T
authors.initials: CD
authors.lastname: Freitas
authors.affiliation: Departamento de Biologia, Centro de Ciências, Universidade Federal do Ceará, Fortaleza, Ceará, Brazil. Electronic address: tbgrangeiro@gmail.com.
authors.forename: Thalles Barbosa
authors.initials: TB
authors.lastname: Grangeiro
mesh_terms: D000465:Algorithms; D000595:Amino Acid Sequence; D000935:Antifungal Agents / Q000737:chemistry / Q000302:isolation & purification* / Q000494:pharmacology*; D001483:Base Sequence; D031107:Calotropis / Q000737:chemistry*; D005670:Fusarium / Q000187:drug effects*; D007840:Latex / Q000737:chemistry; D018625:Microscopy, Atomic Force; D008969:Molecular Sequence Data; D010940:Plant Proteins / Q000737:chemistry* / Q000302:isolation & purification* / Q000494:pharmacology; D013172:Spores, Fungal / Q000187:drug effects*; D013762:Tetrahydrofolate Dehydrogenase
chemical_list: D000935:Antifungal Agents; D007840:Latex; D010940:Plant Proteins; C006094:R388; C003427:thaumatin protein, plant; D013762:Tetrahydrofolate Dehydrogenase
keywords: Apocynaceae; Calotropis procera; Protein; Sodom apple; Thaumatin-like protein