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
Dm. Rhoads et al., 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, Archives of biochemistry and biophysics, 354(1), 1998, pp. 158-164
URF13 is a mitochondrially encoded protein in the inner mitochondrial
membrane of maize (Zea mays L.) carrying the cms-T cytoplasm. This pro
tein is responsible for Texas-type cytoplasmic sterility and is a liga
nd-gated, pore-forming receptor for the pathotoxins of fungal pathogen
s Bipolaris maydis race T and Phyllosticta maydis. URF13 contains thre
e transmembrane cy-helices, with amphipathic helices II and III likely
involved in pore formation, and is present as oligomers in cms-T maiz
e mitochondria and when expressed in Escherichia coli cells, To study
tertiary and quaternary structures of URF13 oligomers, we employed com
binations 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 in E. coli c
ells 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 doubl
e mutants URF13-C27R/R79C, URF13-C27R/R81C, and URF13-C27R/T82C, indic
ating that the cross-linking was between introduced Cys residues on ad
jacent URF13 molecules. Disulfide bond formation, induced by diamide,
was seen only in URF13-T82C and URF13-C27R/T82C, indicating that Cys r
esidues introduced into position 82 are closely juxtaposed in the olig
omers, Based on these observations, we modified the models for the sec
ondary structure of URF13 and the tertiary structure of the URF13 olig
omers. Sequential cross-linking of URF13-R81C oligomers with bismaleim
idohexane (Cys-Cys crosslinker) and N,N'-dicyclohexylcarbodiimide (Lys
-Asp/ Glu cross-linker) suggests that URF13 oligomers consist of an ev
en number of monomers. (C) 1998 Academic Press.