pH-induced Collapse of the Extracellular Loops ClosesEscherichia coli Maltoporin and Allows the Study of Asymmetric Sugar Binding

Abstract

LamB (maltoporin) is essential for the uptake of maltose and malto-oligosaccharides across the outer membrane of Escherichia coli. Purified LamB was reconstituted in artificial lipid bilayer membranes forming channels in the permanently open configuration at neutral pH. Almost complete channel closure was observed when the pH on both sides of the membrane was lowered to pH 4. When LamB was added to only one side of the membrane, the cis-side, and the pH was lowered at either side of the membrane, the cis-or the trans-side, the response to pH was asymmetric, suggesting preferential orientation of maltoporin channels and pHdependent closure of only one side of the channel. In experiments with LamB mutants in which major external loops L4, L6, and/or L9 were deleted, we identified the surface-exposed loops L4 and L6 as the cause of pH-mediated closure. The pH dependence of the LamB channel is consistent with the assumption that it inserts in a preferential orientation into the lipid bilayer. About 70 -80% of the reconstituted channels are oriented with the extracellular entrance toward the side to which the protein was added (the cis-side) and with the periplasmic opening on the opposite side (the trans-side). The possibility of closing the channels, which are oriented in the reverse direction by low pH at the trans-side, allowed the deduction of channel asymmetry with respect to carbohydrate binding kinetics. Whereas maltose binding was found to be almost symmetric with respect to the channel orientation, the sucrose and trehalose binding to LamB was asymmetric. The results are discussed in respect to possible physiological function of the pH-dependent closure of maltoporin.The outer membrane protects Gram-negative bacteria against noxious substances such as bile salts or degrading enzymes like proteinases or lipases (1, 2). Water-filled channels, the so-called porins, allow the passage of solutes through this diffusion barrier. Porins are divided in two classes: general diffusion pores, which sort solutes according to their molecular mass, and specific pores, which have a binding site for specific substrates inside the channel and facilitate the diffusion of these substrates through the outer membrane (3, 4). In the last few years the structure of several porins were solved by x-ray crystallography (5-7). Porins are built of three identical polypeptide subunits (monomers). The common structure of the monomers is a barrel formed by 16 or 18 antiparallel ␤-strands connected by small turns on the periplasmic side and by long loops on the extracellular side. In all known porin structures loop 3, between ␤-strands 5 and 6, is folded inside the channel and leads to a decrease in the diameter of the constriction of the channel. The other loops form a protrusion that protects the extracellular entrance of the outer membrane channels.LamB (maltoporin) from Escherichia coli is a specific porin for malto-oligosaccharides (8, 9). The monomer is composed of 18 ␤-strands, which means that there are nine extracellular loops. Th...

Probing the Orientation of Reconstituted Maltoporin Channels at the Single-protein Level

“…In agreement with the orientation of maltoporin channels suggested by the kinetics of maltohexaose transport through the mutants W74A and F227A, we conclude that maltoporin inserts with the periplasmic turns leaving the extracellular loops oriented at the cis-side of the membrane (Fig. 8), confirming the proposal of Benz and co-workers (21,22). It is noteworthy that Van Gelder et al (23) found an opposite orientation of maltoporin in lipid bilayers.…”

“…Based on the binding asymmetry of a pseudooligosaccharide modified at its nonreducing end (20), on mutants deleted in the large extracellular loops, and on asymmetrical pH-induced closure of the channel proteins, Benz and co-workers (21,22) propose that maltoporin preferentially inserts with the short periplasmic turns moving through the membrane. In contrast, Van Gelder et al (23) used bacteriophage-and observed the opposite orientation.…”

The Core of the Tetrameric Mycobacterial Porin MspA Is an Extremely Stable β-Sheet Domain

“…In principle, this could be due to either a switch of MspA to a closed channel conformation or to a reduced reconstitution efficiency. Voltage-and pH-dependent conformational changes of extracellular loops leading to channel closure were demonstrated for the general porin OmpF (47) and for the maltose-specific porin LamB (48). Since substantial extracellular domains were observed in electron microscopy pictures of MspA in the cell wall of M. smegmatis (12), it seems more likely that the urea-or SDS-induced loss of channel activity reflects structural alterations of MspA rather than a reduced reconstitution efficiency of MspA in lipid bilayer experiments.…”

Alternative Splicing in Class V Myosins Determines Association with Rab10

“…Membranes were formed either from a 1% solution of diphytanoyl phosphatidylcholine (Avanti Polar Lipids, Alabaster, AL) or from a 1% solution of a mixture of diphytanoyl phosphatidylcholine and cholesterol (molar ratio 1:1) or from a 1% solution of oxidized cholesterol (36) in n-decane. Reduction of pH was achieved by the addition of predetermined amounts of 0.1% HCl to the volume on one or both sides bathing lipid bilayer membranes (37).…”