Abstract
The Staphylococcus epidermidis glucose/H+ symporter (GlcPSe) is a membrane transporter highly specific for glucose and a homolog of the human glucose transporters (GLUT, SLC2 family). Most GLUTs and their bacterial counterparts differ in the transport mechanism, adopting uniport and sugar/H+ symport, respectively. Unlike other bacterial GLUT homologs (for example, XylE), GlcPSe has a loose H+/sugar coupling. Asp22 is part of the proton-binding site of GlcPSe and crucial for the glucose/H+ co-transport mechanism. To determine how pH variations affect the proton site and the transporter, we performed surface-enhanced IR absorption spectroscopy on the immobilized GlcPSe We found that Asp22 has a pKa of 8.5 ± 0.1, a value consistent with that determined previously for glucose transport, confirming the central role of this residue for the transport mechanism of GlcPSe A neutral replacement of the negatively charged Asp22 led to positive charge displacements over the entire pH range, suggesting that the polarity change of the WT reflects the protonation state of Asp22 We expected that the substitution of the residue Ile105 for a serine, located within hydrogen-bonding distance to Asp22, would change the microenvironment, but the pKa of Asp22 corresponded to that of the WT. A167E mutation, selected in analogy to the XylE, introduced an additional protonatable site and perturbed the protonation state of Asp22, with the latter now exhibiting a pKa of 6.4. These studies confirm that Asp22 is the proton-binding residue in GlcPSe and show that charged residues in its vicinity affect the pKa of glucose/H+ symport.
Highlights
The cellular uptake of monosaccharides, polyols, and other small carbon compounds across the membranes of eukaryotic cells is an essential physiological process. Their transport is facilitated by specialized transporters that are members of the GLUT family (SLC2 gene family), which belongs to the major facilitator superfamily (MFS), one of the largest protein families with over 10,000 members [1]
The difference spectra reflect structural reorganization within GlcPSe caused by the shift in pH, including changes in the protein backbone conformations and the protonation state of individual side chains; for example, the COOH vibrational mode of protonated acidic residues is typically ;1750 cm21 [24, 25]
The difference spectra for the same pH step of D22A GlcPSe (Fig. 2B) is almost identical to the WT one, except for the important signal at 1750 cm21, confirming that this signal arises from Asp22, a residue previously suggested to play a similar role to Glu325 in LacY [23, 26]
Summary
The cellular uptake of monosaccharides, polyols, and other small carbon compounds across the membranes of eukaryotic cells is an essential physiological process. In humans, their transport is facilitated by specialized transporters that are members of the GLUT family (SLC2 gene family), which belongs to the major facilitator superfamily (MFS), one of the largest protein families with over 10,000 members [1]. MFS proteins presumably share an alternating access mechanism of transport in which the substrate-binding site alternatively opens to either side of the membrane [12]. The crystal structure of the Staphylococcus epidermidis glucose/H1 symporter, a bacterial GLUT homolog, was solved in the inward-facing conformation [8]. Crystal structures of XylE obtained at acidic (pH 5.8) and alkaline (pH 9.6) pH were in the inwardand outward-facing conformations, respectively, with Asp (the equivalent of Asp in GlcPSe) interacting with Arg133 (the equivalent of Arg103 in GlcPSe) in the outward-facing conformation but not in the inward-facing conformation [11, 20]
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