Structure-function analysis of the vitamin B12 receptor of Escherichia coli by means of informational suppression.
We describe a genetic analysis of the vitamin B12 receptor of Escherichia coli. Through the use of informational suppression, we have been able to generate a family of receptor variants, each identical save for a single, known substitution (Ser, Gln, Lys, Tyr, Leu, Cys, Phe) at a known site. We have studied 22 different mutants, 14 in detail, distributed throughout the length of the btuB gene. Most amino acid substitutions have a pleiotropic effect with respect to all ligands tested, the two colicins E1 and E3, the T5-like bacteriophage BF23, and vitamin B12. (The dramatic effect of a single amino acid substitution is also well exemplified by the G142A missense change which renders the receptor 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 membrane (devised using both amphipathic beta-strand analysis and sequence conservation amongst the TonB-dependent receptors). In addition, we report that the extreme C-terminus of BtuB is vital for receptor localization and provide evidence for it being a membrane-spanning beta-sheet with residue L588 situated on its hydrophobic surface. Two of the C-terminal btuB mutations are located within the region of overlap with the recently identified dga (murl) gene.
Hufton
S E
SE
Department of Biochemistry, University of Nottingham Medical School, Queen's Medical Centre, UK.
Ward
R J
RJ
Bunce
N A
NA
Armstrong
J T
JT
Fletcher
A J
AJ
Glass
R E
RE
D000595:Amino Acid Sequence; D001425:Bacterial Outer Membrane Proteins / Q000737:chemistry / Q000235:genetics / Q000502:physiology*; D001426:Bacterial Proteins / Q000502:physiology; D003062:Codon / Q000235:genetics; D003087:Colicins / Q000494:pharmacology; D003090:Coliphages / Q000502:physiology; D004926:Escherichia coli / Q000235:genetics* / Q000378:metabolism; D029968:Escherichia coli Proteins; D005798:Genes, Bacterial; D008024:Ligands; D008565:Membrane Proteins / Q000502:physiology; D026901:Membrane Transport Proteins; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010641:Phenotype; D011487:Protein Conformation; D017510:Protein Folding; D018000:Receptors, Peptide / Q000737:chemistry / Q000187:drug effects / Q000235:genetics / Q000502:physiology*; D012486:Salmonella typhimurium / Q000235:genetics; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid; D013045:Species Specificity; D013329:Structure-Activity Relationship; D013489:Suppression, Genetic; D014805:Vitamin B 12 / Q000378:metabolism
D001425:Bacterial Outer Membrane Proteins; D001426:Bacterial Proteins; C044592:BtuB protein, E coli; D003062:Codon; D003087:Colicins; D029968:Escherichia coli Proteins; D008024:Ligands; D008565:Membrane Proteins; D026901:Membrane Transport Proteins; D018000:Receptors, Peptide; C057616:tonB protein, Bacteria; C489181:tonB protein, E coli; D014805:Vitamin B 12