Cross-Linking and Disulfide Bond Formation of Introduced Cysteine Residues Suggest a Modified Model for the Tertiary Structure of URF13 in the Pore-Forming Oligomers

URF13 is a mitochondrially encoded protein in the inner mitochondrial membrane of maize (Zea maysL.) carrying thecms-Tcytoplasm. This protein is responsible for Texas-type cytoplasmic sterility and is a ligand-gated, pore-forming receptor for the pathotoxins of fungal pathogensBipolaris maydisrace T andPhyllosticta maydis.URF13 contains three transmembrane α-helices, with amphipathic helices II and III likely involved in pore formation, and is present as oligomers incms-Tmaize mitochondria and when expressed inEscherichia colicells. To study tertiary and quaternary structures of URF13 oligomers, we employed combinations of site-directed mutagenesis and chemical cross-linking. We introduced Cys residues individually into consecutive positions 78–82, believed to be in helix III. We expressed these proteins inE. colicells and tested for cross-linking through disulfide bond formation or by using Cys–Cys cross-linkers. URF13-R79C, URF13-R81C, and URF13-T82C were cross-linked using Cys–Cys-specific cross-linkers, as were double mutants URF13-C27R/R79C, URF13-C27R/R81C, and URF13-C27R/T82C, indicating that the cross-linking was between introduced Cys residues on adjacent URF13 molecules. Disulfide bond formation, induced by diamide, was seen only in URF13-T82C and URF13-C27R/T82C, indicating that Cys residues introduced into position 82 are closely juxtaposed in the oligomers. Based on these observations, we modified the models for the secondary structure of URF13 and the tertiary structure of the URF13 oligomers. Sequential cross-linking of URF13-R81C oligomers with bismaleimidohexane (Cys–Cys cross-linker) andN,N′-dicyclohexylcarbodiimide (Lys–Asp/Glu cross-linker) suggests that URF13 oligomers consist of an even number of monomers.