Interaction of transmembrane helices in ATP synthase subunit a in solution as revealed by spin label difference NMR

Abstract

Subunit a in the membrane traversing F0 sector of Escherichia coli ATP synthase is known to fold with five transmembrane helices (TMHs) with residue 218 in TMH IV packing close to residue 248 in TMH V. In this study, we have introduced a spin label probe at Cys residues substituted at positions 222 or 223 and measured the effects on the Trp epsilon NH indole NMR signals of the seven Trp residues in the protein. The protein was purified and NMR experiments were carried out in a chloroform-methanol-H2O (4:4:1) solvent mixture. The spin label at positions 222 or 223 proved to broaden the signals of W231, W232, W235 and W241 located at the periplasmic ends of TMH IV and TMH V and the connecting loop between these helices. The broadening of W241 would require that the loop residues fold back on themselves in a hairpin-like structure much like it is predicted to fold in the native membrane. Placement of the spin label probe at several other positions also proved to have broadening effects on some of these Trp residues and provided additional constraints on folding of TMH IV and TMH V. The effects of the 223 probes on backbone amide resonances of subunit a were also measured by an HNCO experiment and the results are consistent with the two helices folding back on themselves in this solvent mixture. When Cys and Trp were substituted at residues 206 and 254 at the cytoplasmic ends of TMHs IV and V respectively, the W254 resonance was not broadened by the spin label at position 206. We conclude that the helices fold back on themselves in this solvent system and then pack at an angle such that the cytoplasmic ends of the polypeptide backbone are significantly displaced from each other.

Fig. 1

Topological model for folding of subunit a in E. coli inner membrane. The biochemical evidence for the insertion of the five TMHs is discussed in the text. The depth of placement of the helices in the membrane is based upon cross-linking studies as described elsewhere [29]. The positions of the seven Trp residues in the wild type protein are highlighted. The helical segments shown in regions peripheral to the lipid bilayer were predicted by backbone chemical shift analysis for the protein dissolved in chloroform-methanol-water solvent [32].

Fig. 2

Examples of the assignment of the ε-imino indole signal of Trp residues by comparing the spectra of wild type and single W→A substituted mutants. The positions of Trp signals in the spectrum of wild type protein are indicated by the labeled arrows.

Fig. 3

Line broadening of Trp ε-imino indole signals by PROXYL spin label attached at Cys223 of the subunit a. (A) Control spectrum of wild type protein recorded after reducing the PROXYL spin label with phenylhydrazine. Resonance assignments are indicated by the arrows. (B) PROXYL labeled wild type sample showing loss of W231, W232, W235 and W241 resonances. (C) Overlay of the spectra of the spin-labeled W186F variant of subunit a (black) and of the phenylhydrazine reduced sample [red].

Fig. 4

Spin label sites [magenta] tested using Trp ε-imino indole signals as reporter groups. The structure shown is a representative conformer from the ensemble of ten lowest energy structures calculated from the results in this paper. Side chains of Trp residues in the wild type protein are shown in blue. Trp residues introduced by mutagenesis are shown in gray.

Fig. 5

Line broadening of Trp ε-imino indole signals by PROXYL spin label attached at positions 218 (A,B), 222 (C,D) and 249 (E,F) of subunit a. Spectra of the spin labeled proteins are shown on the left (A,C,E) and the control spectra of the samples reduced with phenylhydrzine on the right (B,D,F). Resonance assignments are indicated by the arrows.

Fig. 6

Line broadening of the backbone amide signals in the HNCO experiment recorded with spin labeled protein (left, A,C) and with the same sample after phenylhydrazine reduction (right, B,D). The upper pair of slices (A,B) is taken at the ¹⁵N shift of 121.5 p.p.m. and the lower pair (C,D) at 118.7 p.p.m. Assigned resonances are labeled.

Fig. 7

Effect of PROXYL spin labeling at Cys223 on backbone amide signals in HNCO spectrum for the C-terminal 71 residues of subunit a. The ratio of peak volumes (V_ox/V_red) before and after reduction of the spin label with phenylhydrazine is plotted versus residue number. The peak volumes of residues marked X could not be calculated confidently because of signal overlap, peak splitting, or significant chemical shift changes caused by the addition of phenylhydrazine. The ratio of peak volumes for residue 264 was 1.98 as indicated by the *. The brackets indicate the TMHs predicted in Fig. 1.

Fig. 8

Residues in TMHs IV and V showing significant backbone amide broadening in the HNCO and 2D ¹H, ¹⁵N-TROSY experiments with PROXYL labeled I223C subunit a. The structure shown is a representative conformation from the ensemble of ten lowest structures calculated. Residues are color coded based upon the ratio of amide signal in the spin-labeled versus reduced sample. Oxidized/reduced ratios (V_ox/V_red) and color code are: red = <0.2, orange = 0.2–0.5, yellow= 0.5–0.7, cyan > 0.7. Ratios for the gray residues could not be calculated.

Fig. 9

Modeled structure of the helix IV–helix V segment of subunit a showing ensemble of the 10 lowest energy structures. The r.m.s.d. for the backbone atoms of residues 206–267 fit to a mean structure is 3.6 Å.