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

Abstract

We have directly measured the stoichiometry of maltodextrin-binding sites in LamB. Scatchard plots and computer fitting of flow dialysis (rate-of-dialysis) experiments clearly establish three independent binding sites per LamB trimer, with a dissociation constant of approximately 60 ,uM for maltoheptaose. The current model for LamB's function as a specific pore is discussed with respect to the symmetry in LamB's kinetic properties and the implications of our results.LamB (also called maltoporin) is a trimeric outer membrane protein that functions as a pore for the passage of maltose and maltodextrins [linear a(1-4)-linked D-glucose polymers] across the outer membrane of Escherichia coli (10,24,25,27). A variety of experiments have shown that LamB acts as a passive pore with a binding site for its substrates (2,3,7,8,13,18,19). Binding of maltodextrins inhibits pore activity and has allowed measurements of LamB's binding affinity for maltose and longer maltodextrins. In whole cells, LamB has been shown to act as a saturable pore for certain chromogenic substrate analogs with a measurable Km and Vmax (14).The number of channels (and maltodextrin-binding sites) per LamB trimer has widely been presumed to be either one or three. 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. MATERIALS AND METHODSFlow dialysis. 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.The bottom chamber (volume, -100 RI) was constantly flushed with buffer at 1.1 ml/min, and fractions were collected every minute. Both upper and lower chambers were constantly stirred. The radioactivity in the fractions was determined by liquid scintillation counting and used as a measure of the free [1-3H]maltodextrin concentration. Unlabeled maltodextrin was added at 5-min intervals to the * Corresponding author. The results of the flow dialysis experiments were analyzed by two methods. The first, Scatchard plot analysis, required estimating the rate of [1-3H]maltodextrin elution in the absence of LamB binding sites (the "100% free" rate). In general, at the highest plateau (lowest specific activity), most of the [1-3H]maltodextrin is blocked from the LamB binding site...

Structure and function of bacterial outer membrane proteins: barrels in a nutshell

“…Liposome swelling assays showed that maltoporin has a high permeation rate for maltose and maltodextrins, while uptake of sucrose could hardly be detected (Luckey and Nikaido, 1980). Maltodextrins up to maltoheptaose could be transported, as was shown in in vivo and in vitro uptake experiments with radiolabelled sugars (Gehring et al ., 1991; Charbit et al ., 1998). In contrast, uptake of sucrose is mediated by the plasmid‐borne sucrose regulon, which consists of the scrKYABR genes.…”

pH-Dependent Catabolic Protein Expression during Anaerobic Growth of Escherichia coli K-12

“…Besides enzymes, two sugar transporters were induced at high pH, the maltose oligosaccharide porin MalB (4,17,36) and the galactose binding protein MglB (25). These transporters should be useful for uptake of the hydrolyzed glycogen and lactose in LBK.…”

Noise analysis of ion current through the open and the sugar-induced closed state of the LamB channel of Escherichia coli outer membrane: evaluation of the sugar binding kinetics to the channel interior

“…channel conductance, which has occasionally been observed in noise experiments (Kolb and Bamberg, 1977). On the other hand, it is likely that the LamB trimers contain three identical channels (Ferenci and Lee, 1989;Gehring et al, 1991). Table 3 contains also the stability constants, K*, for maltopentaose binding derived from two parameters fits of So as a function of the maltopentaose concentration similar…”

“…However, the most convincing reason for the small value of g is probably caused by the architecture of LamB in E. coli outer membrane. LamB is organized as trimers in the outer membrane (Palva and Westermann, 1979) and one trimer contains three channels surrounded by a ,3-sheet structure (Clement and Hofnung, 1981;Ferenci et al, 1988;Ferenci and Lee, 1989) and three binding sites for sugars (Gehring et al, 1991). Thus, it is very likely that in the single-channel records three channels are measured simultaneously (g = 150 pS), while the noise analysis recognises the single conductive unit (g = 50 pS).…”