Channel architecture in maltoporin: dominance studies with lamB mutations influencing maltodextrin binding provide evidence for independent selectivity filters in each subunit

Abstract

Maltoporin trimers constitute maltodextrin-selective channels in the outer membrane of Escherichia coli. To study the organization of the maltodextrin-binding site within trimers, dominance studies were undertaken with maltoporin variants of altered binding affinity. It has been established that amino acid substitutions at three dispersed regions of the maltoporin sequence (at residues 8, 82, and 360) resulted specifically in maltodextrin-binding defects and loss of maltodextrin channel selectivity; a substitution at residue 118 increased both binding affinity and maltodextrin transport. Strains heterodiploid for lamB were constructed in which these substitutions were encoded by chromosomal and plasmid-borne genes, and the relative level of maltoporin expression from these genes was estimated. Binding assays with bacteria forming maltoporin heterotrimers were performed in order to test for complementation between binding-negative alleles, negative dominance of negative over wild-type alleles, and possible dominance of negatives over the high-affinity allele. Double mutants with mutations affecting residues 8 and 118, 82 and 118, and 118 and 360 were constructed in vitro, and the dominance properties of the mutations in cis were also tested. There was no complementation between negatives and no negative dominance in heterotrimers. The high-affinity mutation was dominant over negatives in trans but not in cis. The affinity of binding sites in heterotrimer populations was characteristic of the high-affinity allele present and uninfluenced by the negative allele. These results are consistent with the presence of three discrete binding sites in a maltoporin trimer and suggest that the selectivity filter for maltodextrins is not at the interface between the three subunits.

Stoichiometry of maltodextrin-binding sites in LamB, an outer membrane protein from Escherichia coli

“…In agreement with a genetic study of negative dominance and complemention between mutant LamB proteins, we find three binding sites per LamB trimer with no evidence of cooperativity between binding sites on the same trimer (11). The lack of cooperativity and the concordance of our results with those from black lipid studies reinforces the current interpretation of LamB as a simple channel with a single binding site (2,14,18,19).…”

“…This is partially based on images of negatively stained two-dimensional crystals of LamB that show three stain-filled channels merging into one (17). Genetic studies of dominance between mutant LamB proteins have suggested that each LamB monomer forms a single independent pore (11). Here, we confirm the results of this genetic study and show that maltodextrin binding to LamB can be detected in solution and the stoichiometry of binding can be measured.…”

“…Flow dialysis (rate of dialysis) was carried out as described by Colowick and Womack (6), using a homemade dialysis apparatus consisting of two chambers separated by a dialysis membrane. The upper chamber was loaded with 1 ml of concentrated, purified LamB (between 11 and 35 mg/ml) in a solution containing 50 mM Tris * Cl, 100 mM NaCl, 5 mM EDTA, 3 mM NaN3, 10 mM MgCl2, and 0.5% sodium dodecyl sulfate (SDS), pH 7.5. To start the experiment, -50 ,uCi of high-specific-activity [1-3H]maltodextrin (2.5 to 8 Ci/mmol) was added to the upper chamber.…”

A single amino acid substitution alters conductance and gating of OmpC porin ofEscherichia coli

“…Studies aimed at defining functional domains of these nonspecific porins, as well as of maltoporin LamB, have been carried out by the use of mutations in defined regions of the proteins (Heine et al, 1988;Misra & Benson, 1988a,b;Ferenci & Lee, 1989) and by the construction of hybrid proteins (Nogami, Mizuno & Mizushima, 1985;Tommassen et al, 1985;van der Ley et al, 1987, Benz et al, 1989. LamB cells grow poorly on maltose and oligosaccharides, which are presumably too bulky to enter efficiently the cell via the nonspecific OmpF and OmpC porins.…”

The yeast mitochondrial ADP/ATP carrier functions as a monomer in mitochondrial membranes

“…This approach is based on dimers becoming dysfunctional when one protomer is disabled. It has been used to show that lactose permease LacY functions as a monomer (30), that the small drug transporter EmrE functions as an oligomer (31), and that maltoporin functions as a trimer with three distinct selectivity filters (32,33). To establish the functional interaction between monomers, it must be possible to inactivate one protomer and to leave the second monomer unmodified and active.…”