Solution Structure of the Transmembrane H⁺-Transporting Subunit c of the F₁F_o ATP Synthase

Abstract

Subunit c is the H+-translocating component of the F1F0 ATP synthase complex. H+ transport is coupled to conformational changes that ultimately lead to ATP synthesis by the enzyme. The properties of the monomeric subunit in a single-phase solution of chloroform-methanol-water (4:4:1) have been shown to mimic those of the protein in the native complex. Triple resonance NMR experiments were used to determine the complete structure of monomeric subunit c in this solvent mixture. The structure of the protein was defined by >2000 interproton distances, 64 (3)JN alpha, and 43 hydrogen-bonding NMR-derived restraints. The root mean squared deviation for the backbone atoms of the two transmembrane helices was 0.63 A. The protein folds as a hairpin of two antiparallel helical segments, connected by a short structured loop. The conserved Arg41-Gln42-Pro43 form the top of this loop. The essential H+-transporting Asp61 residue is located at a slight break in the middle of the C-terminal helix, just prior to Pro64. The C-terminal helix changes direction by 30 +/- 5 degrees at the conserved Pro64. In its protonated form, the Asp61 lies in a cavity created by the absence of side chains at Gly23 and Gly27 in the N-terminal helix. The shape and charge distribution of the molecular surface of the monomeric protein suggest a packing arrangement for the oligomeric protein in the F0 complex, with the front face of one monomer packing favorably against the back face of a second monomer. The packing suggests that the proton (cation) binding site lies between packed pairs of adjacent subunit c.

Structural and Functional Characterization of Transmembrane Segment IV of the NHE1 Isoform of the Na+/H+ Exchanger

“…Therefore, it is reasonable to presume that they would also be favorable within the setting of the NHE1 protein. Indeed, a number of studies have demonstrated strong correspondence between structures of peptides or protein segments obtained in membrane mimetic solvents to structures obtained by solution state NMR in micelles or to entire membrane proteins determined by x-ray crystallography (11)(12)(13). Most interestingly, we find that CD 3 OH:CDCl 3 :H 2 O provides a well defined peptide structure, whereas the peptide in Me 2 SO appears much less structured.…”

“…This provides insight into structured regions, in our case, without fixing the segment as a whole into a single conformation. The use of solution conditions having a low dielectric constant to mimic a membrane environment has become quite common for structural studies of transmembrane proteins or peptides and has provided structures of isolated protein segments consistent with their structures in the full protein (11)(12)(13). Our study demonstrates that whereas TM IV is structured, only a 4 -6-residue stretch of the segment is helical.…”

NMR investigations of subunit c of the ATP synthase from Propionigenium modestum in chloroform/methanol/water (4 : 4 : 1)

“…Helix I comprises residues L5 to I26, and helix II comprises the segment G29 to N82 with a short interruption between Q46 and P47. In particular no signal intensities were obtained for the a helix characteristic connectivities d aN (23,26), d ab (23,27), d aN (24,27), d ad (24,28), d aN (25,29), d ab (25,28), d ad (25,28) and d aN (26,30). The interruption between helix I and helix II is further confirmed by the small 13 C a downfield chemical shift deviation of the segment A24 to G29.…”

Stochastic rotational catalysis of proton pumping F-ATPase

“…The b subunit is a long helical protein whose aminoterminal is embedded in the membrane, and its carboxyl terminus interacts with a and d at the top of F 1 (Fillingame et al 2000). The structure of the c subunit, two transmembrane a-helices connected by a polar loop, has been solved by NMR (Girvin et al 1998). Low-resolution electron (Birkenhäger et al 1990) and atomic force microscopy (AFM; Singh et al 1996;Takeyasu et al 1996) suggested a ring structure formed from multiple c subunits.…”