Sugar Transport through Maltoporin of Escherichia coli : Role of the Greasy Slide

Abstract

The lining of the maltodextrin-specific maltoporin (LamB) channel exhibits a string of aromatic residues, the greasy slide, part of which has been shown previously by crystallography to be involved in substrate binding. To probe the functional role of the greasy slide, alanine scanning mutagenesis has been performed on the six greasy slide residues and Y118 at the channel constriction. The mutants were characterized by an in vivo uptake assay and sugar-induced-current-noise analysis. Crystallographic analysis of the W74A mutant showed no perturbation of the structure. All mutants showed considerably decreased maltose uptake rates in vivo (<10% of the wild-type value), indicating the functional importance of the investigated residues. Substitutions at the channel center revealed appreciably increased (up to 100-fold) in vitro half-saturation concentrations for maltotriose and maltohexaose binding to the channel. Sugar association rates, however, were significantly affected also by the mutations at either end of the slide (W74A, W358A, and F227A), an effect which became most apparent upon nonsymmetrical sugar addition. The kinetic data are discussed on the basis of an asymmetric one-site two-barrier model, which suggests that, at low substrate concentrations, as are found under physiological conditions, only the heights of the extracellular and periplasmic barriers, which are reduced by the presence of the greasy slide, determine the efficiency of this facilitated diffusion channel.Solute uptake into cells of gram-negative bacteria requires the crossing of two membranes, the outer and the inner membranes, which are separated by the periplasmic space (16). The outer membrane can be considered a defense wall protecting the cell against harmful compounds such as toxins and bile salts. Since a complete seal would impede the import of essential nutrients into the cell, several pore-forming proteins (porins) are located in the outer membrane (19). Transport of solutes through these water-filled channels most commonly proceeds by simple diffusion. For specific porins, diffusional transport is aided by the presence of a binding site.Maltoporin (LamB) specifically facilitates the translocation of malto-oligosaccharides across the outer membrane barrier. Its crystal structure (20) is trimeric, with each monomer consisting of an 18-stranded ␤-barrel enclosing a channel. Long loops protrude into the extracellular space, and short turns face the periplasm. Three loops fold into the ␤-barrel, with loop L3 constricting the channel about halfway through. In the channel lining, there are six contiguous aromatic residues, which form an elongated apolar stripe, the greasy slide, extending from the vestibule through the channel constriction to the periplasmic outlet (Fig. 1). The first of these residues, W74, is provided by L2 of a neighboring subunit. The next three residues, Y41, Y6, and W420, are situated in the middle of the channel. They are followed by W358 and F227, which reside in the wide periplasmic exit hall. A comparable ...

Chitoporin from Vibrio harveyi, a Channel with Exceptional Sugar Specificity

“…This will be a subject of our further investigation. Translocation of chitohexaose particularly involved specific substrate-protein interactions, resulting in Michaelis-Menten transport kinetics resembling those of previously reported sugar-specific porins, including maltoporin (LamB) (31)(32)(33)(34), sucrose porin (ScrY) (25,35), glucoseinducible porin (OprB) (36,37), and cyclodextrin porin (CymA) (38, 39). The on-rate constant (k on ) was estimated from k on ϭ K⅐k off .…”

Docking Studies Show That D-Glucose and Quercetin Slide through the Transporter GLUT1

“…At first sight, this is surprising, given the expectation that glucose should make mainly hydrophilic contacts within GLUT, and hydrophobic amino acids are thought to form nonpolar interactions with the surrounding lipid membrane (41). However, attention has been drawn to the similar high proportion of aromatic residues in the GLUT1 pore (3,31,42) and in maltoporin (LamB) (27,28). Sequence comparison between GLUT1 and maltoporin here shows a number of similarities between the strands lining the ␤-barrel maltoporin and the amino acids lining the hydrophilic pore of GLUT1 (Fig.…”

“…Maltosaccharides are guided through maltoporin by slippage between hydrophobic axial hydrogen atoms projecting from the planar surface of the glycosidic ring and -electrons of aromatic amino acid side chains in one of the maltoporin strands lining the pore. Hydrogen bonds between OH or endocyclic oxygen groups on both sides of the pyranose ring and hydrophilic porin tracks constitute the other two support rails required to guide the maltosaccharide train through the porin (27,28).…”

“…This new model of transport via GLUT1 built on previous transport, structural, and modeling studies (1,2,3,7,10,20,22,(23)(24)(25)(26)(27)(28) delineates how glucose and quercetin may slide via a pore structure and is clearly differentiated from the conventional alternating carrier description of glucose transport. It offers an explanation for the widely dispersed mutation sites affecting GLUT1-DS (30,31).…”

Siderophore Transport through Escherichia coli Outer Membrane Receptor FhuA with Disulfide-tethered Cork and Barrel Domains

“…Highly conserved residues along the inner barrel wall of the putative siderophore transport pathway may facilitate the diffusion of ferrichrome through FhuA by a series of low affinity binding interactions. Such a mechanism was shown for diffusion of maltose and maltodextrins along the greasy slide of LamB porin (10).…”