STRUCTURE-FUNCTION ANALYSIS OF THE VITAMIN-B-12 RECEPTOR OF ESCHERICHIA-COLI BY MEANS OF INFORMATIONAL SUPPRESSION

We describe a genetic analysis of the vitamin B-12 receptor of Escheri chia coli. Through the use of informational suppression, we have been able to generate a family of receptor variants, each identical save fo r a single, known substitution (Ser, Gin, Lys, Tyr, Leu, Cys, Phe) at a known site, We have studied 22 different mutants, 14 in detail, dist ributed throughout the length of the btuB gene, Most amino acid substi tutions have a pleiotropic effect with respect to all ligands tested, the two colicins El and E3, the T5-like bacteriophage BF23, and vitami n B-12- (The dramatic effect of a single amino acid substitution is al so well exemplified by the G142A missense change which renders the rec eptor completely non-functional.) In some instances, however, we have been able to modify a subset of receptor functions (viz, Q62, Q150 and Q299 and the response to phage BF23). These data are summarized on a two-dimensional folding model for the BtuB protein in the outer membra ne (devised using both amphipathic beta-strand analysis and sequence c onservation amongst the TonB-dependent receptors), In addition, we rep ort that the extreme C-terminus of BtuB is vital for receptor localiza tion and provide evidence for it being a membrane-spanning P-sheet wit h residue L588 situated on its hydrophobic surface. Two of the C-termi nal btuB mutations are located within the region of overlap with the r ecently identified dga (murl) gene.