Combinatorial mutagenesis analysis of residues in the channel constriction loop L3 and neighbouring beta-strands in the LamB glycoporin of Escherichia coli.
Members of the LamB family of sugar-selective porins (glycoporins) are beta-barrel proteins in the outer membrane of Gram-negative bacteria. To study the determinants of structure and sugar selectivity, 68 non-identical single amino acid substitutions were introduced into the stretch of sequence consisting of residues 106 through 125 in Escherichia coli LamB. This region includes all bar one residue of the channel constriction loop L3 and extends into the transmembrane beta 6 strand in the LamB structure. Mutants were assayed for dextrin utilization, starch binding, A binding, monoclonal antibody binding and for qualitative changes in protein expression. The importance of the L3 amino acids was emphasized by the observation that only four residues permitted a majority of neutral substitutions. Changes to the channel constriction zone strongly affected sugar binding yet no single amino acid change of residues exposed to the channel lumen caused a complete defect in maltodextrin utilization (i.e. were still Dex+). Substitutions in the L3 loop did not affect phage lambda binding, except one change at residue 122, nor changed recognition by anti-LamB antibodies specific for surface epitopes, consistent with the lack of a role of L3 residues in surface receptor function. In marked contrast, four substitutions in transmembrane strand beta 5 resulted in a Dex- phenotype and gross changes in protein properties, indicating the significance of beta 5 in the architecture of LamB.
Chan
W C
WC
Department of Microbiology, University of Sydney, Australia.
Schirmer
T
T
Ferenci
T
T
D000595:Amino Acid Sequence; D000911:Antibodies, Monoclonal / Q000378:metabolism; D001425:Bacterial Outer Membrane Proteins; D010582:Bacteriophage lambda / Q000378:metabolism; D001665:Binding Sites; D003912:Dextrins / Q000378:metabolism; D004926:Escherichia coli / Q000737:chemistry*; D008969:Molecular Sequence Data; D016296:Mutagenesis; D009154:Mutation; D018272:Porins; D011487:Protein Conformation; D011991:Receptors, Virus / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D013213:Starch / Q000378:metabolism
D000911:Antibodies, Monoclonal; D001425:Bacterial Outer Membrane Proteins; D003912:Dextrins; D018272:Porins; D011991:Receptors, Virus; C016532:maltoporins; D013213:Starch