The fast release of sticky protons: kinetics of substrate binding and proton release in a multidrug transporter

Abstract

EmrE is an Escherichia coli H(+)-coupled multidrug transporter that provides a unique experimental paradigm because of its small size and stability, and because its activity can be studied in detergent solution. In this work, we report a study of the transient kinetics of substrate binding and substrate-induced proton release in EmrE. For this purpose, we measured transient changes in the tryptophan fluorescence upon substrate binding and the rates of substrate-induced proton release. The fluorescence of the essential and fully conserved Trp residue at position 63 is sensitive to the occupancy of the binding site with either protons or substrate. The maximal rate of binding to detergent-solubilized EmrE of TPP(+), a high-affinity substrate, is 2 x 10(7) M(-1).s(-1), a rate typical of diffusion-limited reactions. Rate measurements with medium- and low-affinity substrates imply that the affinity is determined mainly by the k(off) of the substrate. The rates of substrate binding and substrate-induced release of protons are faster at basic pHs and slower at lower pHs. These findings imply that the substrate-binding rates are determined by the generation of the species capable of binding; this is controlled by the high affinity to protons of the glutamate at position 14, because an Asp replacement with a lower pK is faster at the same pHs.

Fig. 1.

Tryptophan fluorescence spectra of EmrE mutants at different pH values with/without substrate. (A–C) Emission spectra (excitation at 280 nm) of 0.5 μM EmrE (A), 0.5 μM E14D (B), and 2 μM single Trp63 (C). The lowest solid line is at pH 5.5, the middle is at pH 7.0, and the highest is at pH 8.5. The dashed line represents protein at pH 8.5 after addition of 50 μM of the substrate TPP⁺. The shoulder at ≈310 nm in the spectra of the single Trp-63 mutant (that bears seven Tyr residues) is not observed upon excitation at 295 nm and was assigned to Tyr fluorescence (14). (D–F) Summary of the pH dependence of the fluorescence intensity at the emission peak at 338 nm of free (squares) and substrate-bound protein (circles).

Fig. 2.

Stopped-flow measurements of tryptophan fluorescence quenching induced by substrate binding at pH 7.0. EmrE (0.5 μM) (A), 2 μM single Trp-63 (B), and 0.5 μM E14D (C) were mixed with 3 or 10 μM TPP⁺. Excitation was at 280 nm, and emission was collected with >320-nm cutoff filter. Each curve is the average of 5–10 repeats, and the data are fitted to an exponential equation. (D) Summary of the observed rates (k_obs = 1/τ) of the binding reaction at increasing TPP⁺ concentrations at pH 7.0. The rate increases linearly, and the slope represents the association constant (k_on) according to Eq. 1.

Fig. 3.

pH dependence of the association constant (k_on). The plot of the k_obs of the binding reaction against the TPP⁺ concentration is presented for EmrE (A), single Trp-63 (B), and E14D (C), at three pH values: 5.5 (squares), 7.0 (circles), and 8.5 (triangles). The slope represents the k_on according to Eq. 1. (D) Summary of the k_on for EmrE (squares), single Trp 63 (open triangles), and E14D (circles) at the pH range from 5.5 to 9.3. The EmrE and E14D data are fitted to the equation for Model C (SI Fig. 10).

Fig. 4.

Parallel measurement of the kinetics of substrate-binding and substrate-induced proton release. (A) Stopped-flow measurements of proton release: 3 μM EmrE in a solution of 0.15 M NaCl, 0.08% DDM buffered with 100 μM phenol red (pH 7.0) was mixed with 3, 6, and 10 μM TPP⁺. Shown also is protein inhibited with DCCD (pretreated with 500 μM, 60 min at room temperature) and protein mixed with TPP⁺ before the experiment. Absorption changes at 556 nm were collected. (B) Same solutions as in A, measured for tryptophan fluorescence changes at 280-nm excitation and >320-nm emission. The data on A and B are an average of 5–10 repeats, fitted to an exponential equation. (C) Summary of the k_on values obtained from measurements like those presented in A and B for 1.5 or 3 μM EmrE at different pH values. The k_on was calculated by using the slope of the linear fit of the k_obs against [TPP⁺] according to Eq. 1. Squares are k_on obtained from substrate-induced proton release measurements, and open circles are k_on obtained from the same solutions measured for substrate binding by using tryptophan fluorescence changes.

Fig. 5.

pH dependence of the equilibrium K_d of EmrE and E14D. Purified protein was immobilized on Ni-NTA beads and bound to increasing [³H]TPP⁺ concentrations at various pH values as described in Materials and Methods. The K_d values are presented for EmrE (squares) and E14D (circles).

Fig. 6.

The substrate-binding reaction cycle in EmrE. E, nonprotonated EmrE; EH₂, protonated EmrE; ES, substrate bound EmrE; E:H₂:S, hypothetical ternary complex of protonated EmrE and substrate.