Aqueous accessibility to the transmembrane regions of subunit c of the Escherichia coli F1F0 ATP synthase

Abstract

Rotary catalysis in F(1)F(0) ATP synthase is powered by proton translocation through the membrane-embedded F(0) sector. Proton binding and release occur in the middle of the membrane at Asp-61 on transmembrane helix (TMH) 2 of subunit c. Previously the reactivity of Cys substituted into TMH2 revealed extensive aqueous access at the cytoplasmic side as probed with Ag(+) and other thiolate-directed reagents. The analysis of aqueous accessibility of membrane-embedded regions in subunit c was extended here to TMH1 and the periplasmic side of TMH2. The Ag(+) sensitivity of Cys substitutions was more limited on the periplasmic versus cytoplasmic side of TMH2. In TMH1, Ag(+) sensitivity was restricted to a pocket of four residues lying directly behind Asp-61. Aqueous accessibility was also probed using Cd(2+), a membrane-impermeant soft metal ion with properties similar to Ag(+). Cd(2+) inhibition was restricted to the I28C substitution in TMH1 and residues surrounding Asp-61 in TMH2. The overall pattern of inhibition, by all of the reagents tested, indicates highest accessibility on the cytoplasmic side of TMH2 and in a pocket of residues around Asp-61, including proximal residues in TMH1. Additionally subunit a was shown to mediate access to this region by the membrane-impermeant probe 2-(trimethylammonium)ethyl methanethiosulfonate. Based upon these results and other information, a pocket of aqueous accessible residues, bordered by the peripheral surface of TMH4 of subunit a, is proposed to extend from the cytoplasmic side of cTMH2 to Asp-61 in the center of the membrane.

FIGURE 1.

Inhibition of ATP-driven proton pumping by Ag⁺. ATP-driven quenching of ACMA fluorescence by inverted membrane vesicles was assayed as described under “Experimental Procedures.” Fluorescence quenching was initiated by the addition of ATP at 20 s and terminated by the addition of nigericin (N) at 100 s. The return to maximum fluorescence after the addition of nigericin was used to calculate the relative quenching values given in Table 1 and the inhibition values given in Fig. 2. The traces are representative of the effect of 40 μm Ag⁺ on vesicles containing no Cys substitutions (A), cA24C (B), cI26C (C), and cI28C (D).

FIGURE 2.

Sensitivity of Cys substitutions to inhibition by Ag⁺. Inhibition after treatment with 40 μm Ag⁺ was calculated from relative quenching values. Each determination is the average of ≥2 trials, and the error bars represent the S.D. Inhibition values for residues 52–65 were taken from Ref. . Cys substitutions at positions 18, 23, 27, 29, 54, 55, and 61 abolished quenching activity. The two helices are aligned vertically based upon the WHATIF model from the crystal structure (8).

FIGURE 3.

Modification of G58C with MTSET in the presence and absence of subunit a. Membrane vesicles containing subunit a (A) or lacking subunit a (B) were treated with 0.5 mm MTSET and, following solubilization with SDS, were then treated with 1 mm NEM as described under “Experimental Procedures.” The MTSET derivatization was then reversed by treatment with β-MSH to generate unmodified subunit c. Subunit c was purified from the SDS-solubilized membrane fraction as described previously (30) and then subjected to electrospray ionization quadrupole ion trap mass spectrometry. Peaks corresponding to unmodified subunit c at 8330 Da and NEM-modified subunit c at 8456 Da are labeled. In the experiment shown in A, unmodified subunit c accounts for 12% of the total subunit c in the experiment shown.

FIGURE 4.

Concentration dependence of Cd²⁺ sensitivity. ATP-driven quenching of ACMA fluorescence by inverted membrane vesicles was assayed as described in Fig. 1. Inhibition by Cd²⁺ present at a range of concentrations was calculated from relative quenching values. The Cys substitutions shown are G58C (▵), I28C (▴), M57C (○), A62C (●), A24C (□), and no Cys (■).

FIGURE 5.

Inhibition of ATP-driven proton pumping by Cd²⁺. ATP-driven quenching of ACMA fluorescence by inverted membrane vesicles was assayed as described in Fig. 1. The return to maximum fluorescence after the addition of nigericin (N) was used to calculate the inhibition values given in Fig. 6. The traces are representative of the effect of 300 μm Cd²⁺ on vesicles containing no Cys substitutions (A), the insensitive I26C substitution (B), the highly sensitive I28C substitution (C), and the moderately sensitive A20P/P64C substitution (D).

FIGURE 6.

Sensitivity of Cys substitutions to inhibition by Cd²⁺. Inhibition after treatment with 300 μm Cd²⁺ was calculated from the relative quenching values. Each determination is the average of ≥2 trials, and the error bars represent the S.D.

FIGURE 7.

Structural modeling of the Ag⁺- and Cd²⁺-sensitive residues in the c-ring. Sensitive residues are highlighted on a model of the E. coli c-ring derived from the x-ray structure of the c-ring from I. tartaricus using the program WHATIF (8). A, TMH2 is shown as a ribbon to reveal the surface of TMH1 that interacts with TMH2. Asp-61 in one of the c subunits is colored cyan. The positions of Ag⁺-sensitive residues in TMH1 of the same c subunit are highlighted in red. B, the outer ring of helices is shown in a surface representation. Asp-61 is colored cyan. The positions of Cd²⁺-sensitive residues showing >75% inhibition are highlighted in red where the 52′, 59′, and 63′ numbers indicate residues packing next to Asp-61 from an adjacent subunit in the c-ring. The vertical positions of Arg-50, Phe-54, Tyr-73, and Met-75 are indicated.

FIGURE 8.

Reagent accessibility at the interface of subunits a and c. The interface of subunit a and subunit c is shown where subunit a is represented by four cylinders corresponding to TMHs 2–5 with the subunit c monomer represented by a model derived from the x-ray structure of the c-ring from I. tartaricus using the program WHATIF (8). The vertical placement of the subunits relative to one another is based on cross-linking between cTMH2 and aTMHs 4–5 (20, 21). Overlapping colored regions indicate sensitivity to Ag⁺ (green), Cd²⁺ (yellow), MTSEA (red), and NEM (purple).