Asp22 drives the protonation state of the Staphylococcus epidermidis glucose/H+ symporter

Abstract

The Staphylococcus epidermidis glucose/H⁺ symporter (GlcP_Se) 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), GlcP_Se has a loose H⁺/sugar coupling. Asp²² is part of the proton-binding site of GlcP_Se 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 GlcP_Se We found that Asp²² has a pK_a 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 GlcP_Se 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 pK_a 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 pK_a of 6.4. These studies confirm that Asp²² is the proton-binding residue in GlcP_Se and show that charged residues in its vicinity affect the pK_a of glucose/H⁺ symport.

Keywords: glucose transport; major facilitator family; major facilitator superfamily; membrane proteins; membrane transport; pK value; proton transport; spectroscopy; surface-enhanced infrared spectroscopy; transport; transport proteins.

Figure 1.

Overall structure of the inward-facing conformation of GlcP_Se showing the mutated positions in the proton-binding site (Protein Data Bank code 4LDS). Asp²² is from helix H1, Ile¹⁰⁵ and Arg¹⁰² are from helix H4, and Ala¹⁶⁷ is from helix H6. The red ball indicates the putative proton-binding site.

Figure 2.

Perfusion-induced FTIR difference spectra of GlcP_Se WT (A) and GlcP_Se D22A (B) obtained from the sample equilibrated at pH 5.5 without sugar subtracted from the sample equilibrated at pH 8.5 (black line) and the reverse subtraction (grey line).

Figure 3.

SEIRA difference spectra of the pH-dependent change of the GlcP_Se WT (A), D22A (B), I105S (C), A167E (D), and A167E/I105G (E). Signal from sample equilibrated at pH 5.5 was subtracted from that equilibrated at pH 7.5 (spectra a), 8.5 (spectra b), and 9.5 (spectra c) in the absence of glucose or the sample equilibrated at pH 5.5 subtracted from that equilibrated at pH 7.5 (spectra d), 8.5 (spectra e), and 9.5 (spectra f) in the presence of glucose.

Figure 4.

Titration curves for the ν(C=O) vibrational mode of Asp²² at 1750 cm⁻¹. A, GlcP_Se WT with (red circle) and without (black circle) glucose and GlcP_Se D22A (blue circle). B, GlcP_Se A167E with (gray circle) and without (green circle) glucose and GlcP_Se I105S with (cyan circle) and without (magenta circle) glucose. The data were triplicated and are shown as means 8.5 ± 0.1. The error bars represent the standard deviation.

Figure 5.

Transient currents recorded at the SSM after a substrate concentration jump, at symmetrical pH as indicated, in GlcP_Se A167E/I105G mutant. A, pH dependence at symmetrical pH as indicated. Transient currents were induced by 30 mm d-glucose at different pH values, as indicated. B, means and S.D. of the respective charge translocations (peak integrals) from at least three data sets, the apparent pK_a is indicated. C, substrate dependence at symmetrical pH as indicated. Sugar-induced currents were measured at pH 8.5. The apparent K_d = 11.4 ± 3.3 mm. The error bars represent the standard deviation.