Zero-length cross-linking of the C-terminal domain of Escherichia coli ribosomal protein L7/L12 to L10 in the ribosome and in the (L7/L12)4-L10 pentameric complex.
L7/L12Cys89 is a variant of L7/L12 that has a single cysteine residue located in the C-terminal domain in which Cys89 is the only cysteine residue in the protein. A cross-link between this site and the single cysteine in L10, residue 70, was formed with 1,4-di[3'-(2'-pyridyldithio)-propionamido]butane, a sulfhydryl-specific homobifunctional reagent of maximum length 16 A. It is now shown that a zero-length disulfide cross-link between L7/L12Cys89 and L10Cys70 is formed by mild oxidation with Cu2+(phenanthroline)3 of either intact ribosomes or the stable, pentameric complex (L7/L12)4-L10. The formation of the zero-length cross-link defines more closely the contact between the two proteins. Protein L10 is located at the base of the L7/L12 stalk where it provides binding sites for the N-terminal domains of both dimers of L7/L12. The L7/L12Cys89-L10Cys70 cross-link lends further support to our previous model that places at least one of the two dimers of L7/L12 on the surface of the body of the 50S subunit in a bent conformation with the C-terminal domain in close proximity to its N-terminal domain, at the base of the L7/L12 stalk. The L7/L12Cys89-L10Cys70 cross-link in the pentameric L8 complex implies that the protein can exist in this bent conformation there as well as in the ribosome.
Makarov
E M
EM
Department of Biological Chemistry, School of Medicine, University of California, Davis 95616.
Oleinikov
A V
AV
Zecherle
G N
GN
Traut
R R
RR
D001665:Binding Sites; D002850:Chromatography, Gel; D002851:Chromatography, High Pressure Liquid; D003432:Cross-Linking Reagents; D003545:Cysteine / Q000737:chemistry*; D004220:Disulfides / Q000737:chemistry; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000737:chemistry*; D015151:Immunoblotting; D011487:Protein Conformation; D000080882:Ribosomal Protein L10; D012269:Ribosomal Proteins / Q000737:chemistry*; D012270:Ribosomes / Q000737:chemistry*; D013439:Sulfhydryl Reagents / Q000737:chemistry
D003432:Cross-Linking Reagents; D004220:Disulfides; D012269:Ribosomal Proteins; D013439:Sulfhydryl Reagents; C016700:ribosomal protein L7-L12; D003545:Cysteine