Locating an extracellular K+-dependent interaction site that modulates betaine-binding of the Na+-coupled betaine symporter BetP

Abstract

BetP, a trimeric Na(+)-coupled betaine symporter, senses hyperosmotic stress via its cytoplasmic C-terminal domain and regulates transport activity in dependence of the cytoplasmic K(+)-concentration. This transport regulation of BetP depends on a sophisticated interaction network. Using single-molecule force spectroscopy we structurally localize and quantify these interactions changing on K(+)-dependent transport activation and substrate-binding. K(+) significantly strengthened all interactions, modulated lifetimes of functionally important structural regions, and increased the mechanical rigidity of the symporter. Substrate-binding could modulate, but not establish most of these K(+)-dependent interactions. A pronounced effect triggered by K(+) was observed at the periplasmic helical loop EH2. Tryptophan quenching experiments revealed that elevated K(+)-concentrations akin to those BetP encounters during hyperosmotic stress trigger the formation of a periplasmic second betaine-binding (S2) site, which was found to be at a similar position reported previously for the BetP homologue CaiT. In BetP, the presence of the S2 site strengthened the interaction between EH2, transmembrane α-helix 12 and the K(+)-sensing C-terminal domain resulting in a K(+)-dependent cooperative betaine-binding.

Fig. 1.

Single-molecule force spectroscopy of BetP. (A) One single BetP protomer from the trimer reconstituted into a membrane of native E. coli lipids was nonspecifically attached to the stylus of an AFM cantilever. Increasing the distance between stylus and membrane establishes a force, which induces stepwise unfolding of BetP. (B) Superimposition of force-distance (F–D) curves recorded upon unfolding of individual BetP protomers being set in an up-regulated state in presence of betaine and K⁺ (400 mM KCl, 100 mM NaCl, 5 mM betaine, 50 mM Tris-HCl, pH 7.5). Every reproducibly occurring fp (increased density) was fitted using the WLC model to reveal the contour length of the unfolded and stretched polypeptide. Numbers at the end of each WLC fit give the numbers of amino acids the stretched polypeptide. Major fp occurring at a probability of > 80% were labeled red. At close proximity to the membrane (< 25 nm) four partially overlapping peaks occurring were detected. All F-D curves superimposed (A) were recorded at a pulling velocity of 100 nm/s. Histograms show probability (B) and average forces (C) of fp detected at certain contour lengths. Blue lines represent Gaussian fits of histograms. n gives the number of F-D curves superimposed and analyzed.

Fig. 2.

Mapping interactions on the secondary structure of BetP. (A) Interactions mapped onto the secondary structure. Red colored aa highlight interactions detected in Fig. 1. aa colored at less intensity approximate the full-width-at-half maximum (FWHM) of the average fp. Numbers at arrows give the structural position of the interaction detected. This structural position corresponds to the N-terminal Strept-tag II (8 aa) subtracted from the average contour length as revealed from the WLC fitting of individual fp (given in brackets). In case the interaction had to be assumed to lie within the membrane or at the opposite of the membrane a certain number of aa were added (indicated by +) to the contour length to structurally locate the interaction. This procedure called “membrane compensation” (16) is described in the SI Text. All 12 transmembrane α-helices (TMHs) of BetP were labeled with bold numerals. The short helices in loop4 and loop9 at the cytoplasmic and periplasmic sides were denoted IH1 and EH2, respectively. The amphiphatic α-helix H7 is thought to face the periplasm and to make contacts with TMH1, TMH2, and TMH9 and H7 of the other two BetP molecules forming the BetP trimer (12). (B) Side view on the BetP trimer (PDB entry code 2WIT) showing two adjacent protomers in cylinder presentation indicating molecular interaction sites detected by SMFS. TMHs are numbered in white and the contour lengths of fp are given in red or black. One protomer is colored in gray with labeled interaction sites in red according to (A). The membrane is shown as gray rectangle. (C) Top view on one protomer from the periplasmic side. (D) Central substrate-binding site of BetP with a betaine molecule in stick presentation colored in black located close to two interaction sites in TMH3 and TMH8.

Fig. 3.

SMFS spectra recorded upon unfolding of BetpΔC45 recorded in buffer solution that enables transporter activation and substrate-binding. (A) Superimpositions of 95 F-D curves. Histograms show probability (B) and average forces (C) of fp detected at certain contour lengths. n gives the number of F-D curves superimposed. F-D curves were recorded in buffer condition (400 mM KCl, 100 mM NaCl, 5 mM betaine, 50 mM Tris-HCl, pH 7.5) at a pulling velocity of 100 nm/s. fp were marked with p-values of < 0.1 (*), < 0.05 (**), and < 0.01 (***) from T-student tests that show which of the fp changes compared to WT BetP (Fig. 1) were significant.

Fig. 4.

Superimposition of F-D curves recorded at buffer conditions setting BetP into different functional states. (A–C) BetP in an up-regulated state in absence of betaine (400 mM KCl, 100 mM NaCl, 50 mM Tris-HCl, pH 7.5), (D–F) BetP in a down-regulated state in presence of betaine and in absence of K⁺ (500 mM NaCl, 5 mM betaine, 50 mM Tris-HCl, pH 7.5), and (G–I) BetP in a down-regulated state in absence of betaine and K⁺ (500 mM NaCl, 50 mM Tris-HCl, pH 7.5). All F-D curves superimposed (A, D, and G) were recorded at a pulling velocity of 100 nm/s. Histograms show probability (B, E, and H) and average forces (C, F, and I) of fp detected at certain contour lengths. Blue lines represent Gaussian fits of histogram and gray lines are Gaussian fits of the reference data shown in Fig. 1. fp were marked with p-values of < 0.1 (*), < 0.05 (**), and < 0.01 (***) from T-student tests that show which of the fp changes compared to BetP characterized in 400 mM KCl, 100 mM NaCl, 5 mM betaine, 50 mM Tris-HCl, at pH 7.5 (Fig. 1 B–D) are significant. Because the comparison of individual fp can vary for certain pulling velocities, the p-values are averages revealed from comparing the fp across all pulling velocities (Tables S1 and S2).

Fig. 5.

Superposition of residues coordinating the second substrate-binding site in CaiT (yellow) with the periplasmic side of BetP. BetP is shown in spiral presentation and colored as in Fig. 2. Only TMH3, TMH8, TMH12, and EH2 of CaiT are depicted and colored in yellow (PDB entry code 2WSW). The periplasmic second substrate-binding site in CaiT is shown in stick presentation with individual residues in yellow and the substrate γ-butyrobetaine in black.

Fig. 6.

Maximum tryptophan fluorescence studies performed for BetP reconstituted in liposomes in the absence and presence of potassium. (A) In the presence of 450 mM K⁺ WT BetP shows a biphasic binding isotherm with apparent binding constants of K_d1 = 0.49 ± 0.09 mM and K_d2 = 1.01 ± 0.04 mM. Inset: Positive cooperativity with a Hill factor of 1.8 ± 0.3. (B) In the absence of K⁺ a single binding event with a Hill factor of 1.0 ± 0.1 and K_d = 0.52 ± 0.03 mM was detected. (C) Tryptophan fluorescence measured for the BetP mutant BetP-W366A/Q519A in proteoliposomes as a function of the external betaine concentration in the presence of 450 mM K⁺. Each data point shows the average of at least three independent experiments. A binding constant of K_d = 39.2 ± 4.3 mM and a Hill factor of 0.9 ± 0.1 show the dramatic effect of the mutation on the periplasmic second betaine-binding (S2) site. S1 and S2 assign primary and secondary substrate-binding sites, respectively. (B) assigns betaine, K⁺ potassium, Na⁺ sodium, and X the mutated and inactivated S2 site.

Fig. P1.

An extracellular K⁺-dependent interaction site modulates betaine binding of the transmembrane Na⁺-coupled betaine symporter BetP. In the Na⁺-coupled betaine symporter BetP (gray shaded molecule) from Corynebacterium glutamicum, the cytoplasmic K⁺-concentration establishes a second periplasmic substrate-binding (S2) site via an interaction (green upper arrow) between transmembrane helix 12 (TM12) and a helical segment (EH2) in loop9. This interaction is affected by K⁺ binding to the cytoplasmic side (green lower arrow) and the C-terminal domain. A betaine molecule (purple molecule at periplasmic side) is modeled in the X-ray structure of BetP (PDB entry code 2WIT) to indicate the location of this potential S2 site. The membrane bilayer is indicated yellow.