Dynamics of the l -fucose/H ⁺ symporter revealed by fluorescence spectroscopy

Abstract

FucP of Escherichia coli catalyzes L-fucose/H + symport, and a crystal structure in an outward-facing conformation has been reported. However, nothing is known about FucP conformational dynamics. Here, we show that addition of L-fucose to purified FucP in detergent induces ∼20% quenching of Trp fluorescence in a concentration-dependent manner without a shift in λ max . Quenching is essentially abolished when both Trp38 and Trp278, which are positioned on opposing faces of the outward-facing cavity walls, are replaced with Tyr or Phe, and reduced quenching is observed when either Trp is mutated. Therefore, both Trp residues are involved in the phenomenon. Furthermore, replacement of either Trp38 or Trp278, predominantly Trp38, causes decreased quenching, decreased apparent affinity for L-fucose, and significant inhibition of active L-fucose transport, indicating that the two residues are likely involved directly in sugar binding. It is proposed that sugar binding induces a conformational change in which the outward-facing cavity in FucP closes, thereby bringing Trp38 and Trp278 into close proximity around the bound sugar to form an "occluded" intermediate. The location of these two Trp residues provides a unique method for analyzing structural dynamics in FucP.membrane proteins | membranes | protein dynamics | tryptophan A naturally occurring methylpentose, L-fucose (6-deoxy-Lgalactose) is found in mammalian cells as a major component of N-and O-linked glycans and glycolipids (1, 2). In addition, L-fucose, which is present in bacterial polysaccharides, is needed for adhesion and localization and is also used by a variety of microorganisms as a carbon and energy source (3, 4). In Escherichia coli, transport of L-fucose is mediated by the transmembrane protein FucP, which is encoded by fucP, one of the genes of the fuc regulon that is responsible for the catabolism of L-fucose (5-7). FucP consists of 438 amino acids (47,773 Da) and is an L-fucose/H + symporter belonging to the fucose-galactose-glucose/H + symporter (FGHS) subfamily of the major facilitator superfamily (MFS) (8-10). FucP catalyzes symport (cotransport) of L-fucose and H + across the cytoplasmic membrane by transducing free energy stored in the electrochemical H + gradient into a substrate concentration gradient (5,8,9).FucP shares limited sequence homology with other sugar/H + symporters (9), but the X-ray crystal structure reveals many characteristics observed in other MFS transporters (11). Thus, FucP contains 12 transmembrane α-helical domains arranged into two pseudosymmetrical 6-helix bundles surrounding a deep water-filled cavity with the N and C termini on the cytoplasmic side of the membrane. However, unlike other MFS transporters, FucP is in an outward-facing conformation; the cavity is open on the periplasmic side and tightly sealed on the cytoplasmic side. Although it is likely that FucP functions by an alternating access mechanism like LacY (reviewed in ref. 12), nothing is known about its functional dynamics.Out of a total of six Trp ...

Evolutionary mix-and-match with MFS transporters II

“…6 and 16). Furthermore, X-ray structures (18)(19)(20)(21), as well as spectroscopic findings (22), indicate that other MFS members probably function in similar fashion.…”

Function, Structure, and Evolution of the Major Facilitator Superfamily: The LacY Manifesto

“…Moreover, the inherent symmetry of the proteins with regard to the helix kinks and bends provides further nonspeciffic overlap [39]. Moreover, regions of functional similarity (e.g., substrateand/or H + -binding sites) do not align in less homologous MFS transporters; the functional counterparts are often located in distant parts of the protein (Figures 9(a) and 9(b)) [45,[141][142][143].…”

“…However, with a growing number of crystallographic structures, we can begin to identify functionally related residues, which are not aligned in the primary structures. The crystallographic model of FucP was obtained with the central cavity open to the periplasm [22] (Figure 9(a) completely abolishes L-fucose/H + symport [22], but the mutants exhibit counterflow activity that is at least as good as WT [22], and they bind L-fucose with WT affinity [141]. The observations indicate that, like Glu325 mutants in LacY, Asp46 mutants in FucP are specifically blocked in reactions that do involve H + translocation [101].…”

Evolutionary mix-and-match with MFS transporters

“…Rather, the protein essentially moves around the sugar, alternatively exposing both sites to either side of the membrane (3). A wealth of biochemical and biophysical data is available demonstrating that the alternating access model is operative in LacY [reviewed in Smirnova et al (4)], and initial experiments indicate that fucose permease (FucP), a distantly related MFS member, likely functions in similar fashion (17).…”