Structural analysis of ribosomal protein L7/L12 by the heterobifunctional cross-linker 4-(6-formyl-3-azidophenoxy)butyrimidate

The structure of the dimeric form of the protein L7/L12 from ribosomes from Escherichia coli was studied by using the heterobifunctional cross-linker 4-(6-formyl-3-azidophenoxy)butyrimidate. The imidate group of the cross-linker reacts very specifically with Lys-51 of L7/L12. Subsequent cross-linking of this modified L7/L12 by reductive alkylation of the aldehyde group of the cross-linker results in the formation of a covalent cross-link between both polypeptide chains of the L7/L12 dimer. This covalently cross-linked dimer is fully active in reconstitution of elongation factor G dependent GTP hydrolysis of 50S cores lacking L7/L12, suggesting a conformation of the cross-linked protein similar to the conformation of native L7/L12. Analysis of the tryptic peptides of cross-linked L7/L12 shows the points of attachment of the cross-linker to be Lys-51 in one polypeptide chain and Lys-29 in the other. On the basis of a combination of this result with published data, a structure for the N-terminal region of L7/L12 dimers is proposed. The important feature of this model is a shifted parallel alignment of both polypeptide chains resulting in one free N-terminal stretch for each L7/L12 dimer which attaches the protein to the ribosome via protein L10.