Ion-binding titrations

To determine the affinities of the KdpFABC binding sites for both ion species, K⁺ and H⁺, a set of titration experiments was performed. Starting with 2 mL buffer containing 50 mM Tris-HCl (pH 7.8), 2 mM MgCl₂, after thermal equilibration 200 mM RH421 and 9 µg/mL detergent-solubilized enzyme, and subsequently KCl or HCl was added in appropriately small aliquots from stock-solutions until the fluorescence signal saturated. In this manner the existing binding site(s) were titrated, allowing a determination of the half-saturating concentration for K⁺ or protons, K1/2 and pK, respectively. These experiments were started in the buffer, in which KdpFABC is assumed to be in its unphosphorylated E1

state, or in a phosphorylated state produced by addition of either Mg-ATP or Tris-Pi. All titration experiments were repeated 3-5 times and the corresponding averaged values of normalized fluorescence levels were plotted against pH or K⁺ concentration.

Results shown in Figure 32A represent K⁺ titration in the buffer at pH 7.8, and after addition of 1 mM ATP or 1 mM Pi, when KdpFABC is supposed to adopt the E1 and E2-P conformations, respectively. The K⁺-concentration dependence in the E₁ conformation of the KdpFABC (Figure 32A, full circles), was fitted by a Hill function with half-saturating concentration K_1/2 of (6.5 ± 0.3) µM and a Hill coefficient n of 2.3 ± 0.3 (Figure 32A, solid line). Hill coefficient larger than 2 indicates a cooperative binding of more than one K⁺. No satisfying fit could be obtained using a simple Michaelis-Menten kinetics (Figure 32A, dashed line).

69

Figure 32. Electrogenic binding of K⁺ ions in buffer (E₁ state, noted as Kdp only) and after addition of 1 mM Mg-ATP or 1 mM Pi (P-E2 state). A: K⁺ titrations at pH 7.8 with calculated K1/2 values of 6.5 µM (Kdp only), 14.4 µM (P_i), and 16.8 µM (ATP). B: Similar experiments performed at pH 6.1. The half-saturating concentrations K_1/2 were 7.9 µM (Kdp only), 39.1 µM (P_i), and 31.6 µM (ATP). The lines drawn through the data were fits with either a Hill function of a Michaelis-Menten binding isotherm.

In the second set of experiments, phosphorylation of the KdpFABC was triggered by addition of 1 mM Mg-ATP, converting the enzyme to the P-E2 conformation, followed by a KCl titration. In this case, the K⁺ dependence could be fit with a Michaelis-Menten kinetics, providing the half-saturating concentration K1/2 of (16.8 ± 3.0) µM. In the presence of ATP and KCl, the pump works in its turnover mode, and it is not completely clear if the KdpFABC is preferentially in the P-E2 conformation under these conditions. Therefore, another approach to investigate the K⁺ binding affinity in the phosphorylated state of the

70 enzyme is addition of 1 mM Tris-phosphate, instead of ATP, before the corresponding KCl titration was performed. Under these conditions, KdpFABC is supposed to be trapped in a P-E2 state, similar to behavior already known for other members of P-type ATPases. The results of K⁺-titration experiments in the presence of 1 mM Tris-Pi were also fitted with a simple Michaelis-Menten kinetics (Figure 32A, open circles), providing a half-saturating concentration of (14.4 ± 2.6) µM. Comparing the values obtained for the K⁺-binding affinity for phosphorylated KdpFABC complex, produced by ATP and Pi, no significant difference was observed. This result is not unexpected, since the K⁺ binding kinetics should not depend on the phosphorylation method. On the other hand, from the obtained agreement of the half-saturating concentrations it can be assumed that the enzyme complex adopts preferentially the P-E₂ conformation in both sets of measurements. The apparent K⁺-binding affinity was, however, decreased by a factor of ~2 with the respect to the half-saturation determined in the unphosphorylated state (E₁). Noteworthy is the observation that in all KCl titration experiments, performed under three different conditions, the maximum fluorescence decrease of ~0.04 was nearly identical. This implies that the same number of K⁺ ions bind to the membrane domain of the KdpFABC complex, independently of the conformational state of the enzyme.

To investigate to effect of possible proton binding on K⁺ binding affinity, the titration experiments were repeated at pH 6.1. For adjusting the desired pH HCl was used, prior additions of other substrates. KCl titrations were performed in buffer (E1 state) and P-E2

conformation, achieved by addition of either 1 mM Mg-ATP or 1 mM Tris-Pi (Figure 32B).

The pH measured at the end of experiments did not differ more than ± 0.1 from the initial pH. All three data sets could be fitted by a simple Michaelis-Menten kinetics. In the E1

conformation (Figure 32B, full circles) the calculated K1/2 value of (7.9 ± 0.7) µM, was slightly increased with respect to the value obtained at pH 7.8. This observation indicates that higher proton concentration reduces the K⁺ binding affinity in E1 state. This effect is even more pronounced in the P-E₂ conformation of the enzyme, triggered both by ATP and Pi. In this case, the presence of more protons significantly increased half-saturating concentrations to (31.6 ± 1.1) µM and (39.1 ± 7.3) µM, in the presence of ATP (full squares) and P_i (open circles), respectively. Similar to the behavior observed at pH 7.8, the same number of K⁺ ions is bound inside the membrane domain of the KdpFABC complex since the maximum fluorescence decrease was almost the same in all three cases, and furthermore, almost identical to the decrease produced by K⁺ binding at pH 7.8. The

71 presence of higher H⁺ concentration, however, reduced the apparent K⁺ binding affinity only to a minor extent in the unphosphorylated state, whilst the apparent K⁺ binding affinity in the phosphorylated, P-E2, state was significantly reduced, resulting in approximately twofold K1/2 values.

To further investigate the effect of protons in the KdpFABC pumping cycle, proton titrations were carried out correspondingly under different conditions (Figure 33A). Starting with buffer pH 8.0 - 8.1, aliquots of HCl were added until a pH of 6.1 was achieved. The relative fluorescence changes upon proton titration and the pK values for the proton binding were determined by fitting the pH dependence with the Hill function. Proton titration experiments performed in the E₁ state of the enzyme and in the absence of K⁺ (Kdp only, full circles), were fitted with a pK of 7.55 ± 0.05 and a Hill coefficient of n > 1.4. When proton titration was performed in the P-E₂ state in the absence of K⁺, determined pK values were 6.78 ± 0.20 and 7.34 ± 0.04, when the phosphorylated state was produced by 1 mM Mg-ATP or 1 mM Tris-P_i, respectively. The results presented in Figure 33B show the dependence of the pK values on the KCl concentration up to 500 µM in the unphosphorylated state of the enzyme. These data indicate that the K⁺ concentration in the buffer has no significant effect on the pK value. If K⁺ binding would compete with protons, the apparent pK values should decrease with increasing KCl concentrations. At the highest used KCl concentration of 500 µM, which is significantly higher than saturating K⁺ concentration, the pK value was 7.65 ± 0.20. As for the fluorescence decrease at the lowest tested pH ~ 6 upon H⁺ titration dependence on KCl concentration, similar effect was observed (Figure 33C). It was found that maximal fluorescence decrease was independent on the K⁺ concentration present in the buffer, with ΔFmax(pH~ 6) of -0.057 ± 0.002 at 0 K⁺, and -0.050 ± 0.01 at 500 µM K⁺. Obtained values of the K⁺ and H⁺ binding affinities under various conditions are summarized in Table 3.

Comparing the results compiled in Table 3, it is apparent that the K⁺ binding affinity in the phosphorylated state of the KdpFABC complex is generally lower than in the unphosphorylated conformation. Higher proton concentration reduces the K⁺ binding affinity, and this effect is stronger in the P-E₂ state, achieved either by ATP or P_i. In contrast, there are no significant differences in pK in the absence and presence of K⁺ in all three tested states. Additionally, it was found that pK values were strongly dependent of the phosphorylation method. The pK value in phosphorylated state achieved by addition of ATP was found to be 5.70 ± 0.30, whereas upon back-door phosphorylation it was 7.20 ± 0.20.

72

Figure 33. pH titration experiments with the detergent-solubilized KdpFABC complex in different conformations, in the absence and presence of different K⁺ concentrations. A: In the E₁ conformation (Kdp only) a pK of 7.55 could be determined, whereas in the P-E₂ conformation pK values of 6.78 (+ATP) and 7.34 (+ Pi) were determined. The lines drawn represent fits with the Hill function. B: Distribution of pK values obtained in corresponding pH titrations in the presence of different K⁺ concentrations. The pK increased only slightly from 7.55 (0 KCl) to 7.65 (500 µM KCl). C: pH titration experiments in the presence of different K⁺ concentrations, showing that maximal fluorescence decrease was independent on the K⁺ concentration present in the buffer.

6 7 8

pH

-0.06 -0.04 -0.02 0

fluorescence (norm.)

0 K⁺ 60 µM K⁺ 120 µM K⁺ 500 µM K⁺

C

73 Buffer + 1 mM ATP + 1 mM P_i

K1/2 / µM

pH 7.8 6.5 ± 0.3 16.8 ± 3.0 14.4 ± 2.6

pH 6.1 7.9 ± 0.7 31.6 ± 1.1 39.1 ± 7.3

pK

0 K⁺ 7.55 ± 0.05 6.78 ± 0.28 7.34 ± 0.04 500 µM K⁺ 7.65± 0.20 5.70 ± 0.30 7.20 ± 0.20

Table 3. Comparison of ion-binding properties of detergent-solubilized KdpFABC. Half-saturating K⁺ concentrations were determined at pH 7.8 and pH 6.1 for both the unphosphorylated and phosphorylated state induced by the addition of either Mg-ATP or inorganic phosphate (P_i). Apparent pK values were obtained in the absence and presence of saturating 500 µM KCl.

To investigate this remarkable asymmetry of K⁺ and H⁺ competition, the K⁺ binding kinetics in the E₁ state was analyzed in terms of a Lineweaver-Burk plot. K⁺ titrations at five different pH values in the range of 6.1 – 7.8 are shown in Figure 34. The interception of the linear-regression lines through the data points is above the 1/[K⁺] axis, and it indicates a mixed inhibition of K⁺ binding by protons. This finding suggests that at least two different mechanisms contribute to the inhibitory effect.

Figure 34. Lineweaver-Burk plot of K⁺ binding to solubilized KdpFABC in its E₁ conformation at different pH values. The interception of the regression lines through the data points above the 1/[K⁺] axis indicates a mixed inhibition of K⁺ binding by H⁺.

74 It was already reported that KdpFABC is capable of transporting Na⁺, as well as other cations (90), in the absence of K⁺. To test whether Na⁺ ions are able bind to the K⁺-binding sites, corresponding Na⁺-titration experiments were performed (Figure 35). Starting from 2 mL buffer, containing 50 mM Tris-HCl (pH 7.8), 2 mM MgCl2, 200 nM RH421 and 9 µg/mL detergent-solubilized KdpFABC, appropriate aliquots of NaCl stock-solution were added. The concentration dependence could be fit with a simple Michaelis-Menten kinetics, with half-saturating concentration K1/2(Na⁺) of (4.3 ± 0.2) mM. Interestingly, the maximal fluorescence decrease of ~5 % was nearly identical as in the case of corresponding K⁺ titration, although the binding affinity is lower by a factor of ~660.

Figure 35. Na⁺ titration of the solubilized KdpFABC at pH 7.8 in the absence of K⁺. Data were fitted with a simple Michaelis-Menten binding kinetics, with K_1/2(Na⁺) of (4.3 ± 0.2) mM.