Maltose transport and starch binding in phage-resistant point mutants of maltoporin. Functional and topological...

The relationships between the bacteriophage lambda binding site, the starch binding site and the pore formed by maltoporin (LamB protein, lambda receptor protein) were investigated. Bacteria with single amino acid substitutions in the maltoporin sequence, which were previously shown to be strongly reduced in phage lambda sensitivity, were assayed for maltose- (and maltodextrin) selective pore functions. Maltose transport assays was performed at low substrate concentrations, under conditions where LamB is limiting for transport. It revealed three classes of mutants. Class A is composed of mutants with no effect on transport (substitutions at amino acid residues 154, 155, 259, 382 and 401); class B corresponds to mutants with a significant but variable reduction in transport (sites 148, 151, 152, 163, 164, 245, 247 and 250); class C is represented by a single mutant for which transport is almost completely abolished (site 18). Starch binding was assayed by two different methods that gave compatible results. In class A mutants, binding was normal, while no binding was observed in the class C mutant. Binding was impaired to various extents in category B mutants. There was a correlation between the level of impairment of starch binding and impairment of maltose transport, consistent with the notion that the residues influencing starch binding are inside, or in close proximity to, the pore. These results, together with previous data on starch-binding mutants that were not affected in phage binding (substitutions at residues 8, 74, 82, 118 and 121), suggest that the binding sites for starch and phage lambda overlap but are distinct. Mutations affecting transport and starch binding are located in the first third of the protein and in the region of residues 245 to 250. Mutations affecting phage adsorption are located mainly in the last two-thirds of the protein. The topological constraints suggested by the results with the available mutants altered in the lamB gene were used to propose a revised model of maltoporin folding across the outer membrane as well as to define the outlines of footprints of macromolecular binding sites (phage, starch and monoclonal antibodies) on the surface of the protein.