A cluster of charged and aromatic residues in the C-terminal portion of maltoporin participates in sugar binding and uptake.
The maltoporin LamB of Escherichia coli K12 is a trimeric protein which facilitates the diffusion of maltose and maltodextrins through the bacterial outer membrane, and also acts as a non-specific porin for small hydrophilic molecules as well as a receptor for phages. Loop L9 (residues 375 to 405) is the most distal and largest surface-exposed loop of LamB. It comprises a central portion, which varies in size and sequence in the maltoporins of known sequence, flanked by two conserved regions containing charged and aromatic residues. In order to identify the residues within the proximal region that are specifically involved in sugar utilization, we used site-directed mutagenesis to change, individually, each of the charged (five) and aromatic (three) residues in the region 371 to 379 into alanine. None of the eight single amino acid substitutions affected the phage receptor activity of LamB. In contrast, they all affected, to variable extents, maltoporin functions. For all the mutants, very good correlations were observed between the effects on sugar binding and on in vivo uptake. In no case were maltoporin functions completely abolished. Mutants E374 A and W376 A were the most impaired (with over 60% reduction in dextrin binding and in vivo uptake of maltose and maltopentaose). These two mutations also led to an increased bacterial sensitivity to bacitracin and vancomycin. The functional and structural implications are discussed.
Charbit
A
A
Unité de Programmation Moléculaire et Toxicologie Génétique, CNRS URA1444, Istitut Pasteur, Paris, France. acharbit@pasteur.fr
Wang
J
J
Michel
V
V
Hofnung
M
M
D000595:Amino Acid Sequence; D000900:Anti-Bacterial Agents / Q000494:pharmacology; D001414:Bacitracin / Q000494:pharmacology; D001425:Bacterial Outer Membrane Proteins / Q000235:genetics / Q000378:metabolism; D001435:Bacteriophages / Q000472:pathogenicity; D001692:Biological Transport; D004352:Drug Resistance, Microbial; D004926:Escherichia coli / Q000187:drug effects / Q000821:virology*; D029968:Escherichia coli Proteins; D008320:Maltose / Q000378:metabolism*; D026901:Membrane Transport Proteins; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009844:Oligosaccharides / Q000378:metabolism; D011134:Polysaccharides / Q000378:metabolism; D018272:Porins; D011991:Receptors, Virus / Q000737:chemistry / Q000235:genetics* / Q000276:immunology / Q000378:metabolism*; D014640:Vancomycin / Q000494:pharmacology
D000900:Anti-Bacterial Agents; D001425:Bacterial Outer Membrane Proteins; D029968:Escherichia coli Proteins; D026901:Membrane Transport Proteins; D009844:Oligosaccharides; D011134:Polysaccharides; D018272:Porins; D011991:Receptors, Virus; C016532:maltoporins; C073126:tolC protein, E coli; D001414:Bacitracin; C019193:maltopentaose; D008320:Maltose; D014640:Vancomycin; C008315:maltodextrin