Segment TM7 from the cytoplasmic hemi-channel from VO-H+-V-ATPase includes a flexible region that has a potential role in proton translocation

Abstract

A 900-MHz NMR study is reported of peptide sMTM7 that mimics the cytoplasmic proton hemi-channel domain of the seventh transmembrane segment (TM7) from subunit a of H(+)-V-ATPase from Saccharomyces cerevisiae. The peptide encompasses the amino acid residues known to actively participate in proton translocation. In addition, peptide sMTM7 contains the amino acid residues that upon mutation cause V-ATPase to become resistant against the inhibitor bafilomycin. 2D TOCSY and NOESY (1)H-(1)H NMR spectra are obtained of sMTM7 dissolved in d(6)-DMSO and are used to calculate the three-dimensional structure of the peptide. The NMR-based structures and corresponding dynamical features of peptide sMTM7 show that sMTM7 is composed of two alpha-helical regions. These regions are separated by a flexible hinge of two residues. The hinge acts as a ball-and-joint socket and both helical segments move independently with respect to one another. This movement in TM7 is suggested to cause the opening and closing of the cytoplasmic proton hemi-channel and enables proton translocation.

New insights into structure‐function relationships between archeal ATP synthase (A1A0) and vacuolar type ATPase (V1V0)

“…While the cytosolic orientation of the N-terminal tail is generally accepted, its transmembrane topology remains controversial as recently discussed. (55) So far a six, (57,58) eight, (59,60) and nine (61,62) transmembrane helix topological model of the membraneembedded part of a has been proposed. According to the topology model with eight transmembrane helixes, both N-and the C-terminal tails of a isoforms (Vph1p and Stv1p) are located in the cytosol, which is supported by experimental data, showing interaction of phosphofructokinase-1 with the C-terminal tail of the human a4 and a1 isoforms.…”

“…Cysteine cross-linking experiments demonstrated that helix TM7 of a subunit is in the proximity to TM4 of c and c 0 subunits and to TM3 of c 00 subunit, containing Glu residues, essential for proton translocation during rotation of the c ring. (62) Based on this data, the model of swiveling of interacting helices in a and c of the proteolipid ring was suggested. This swiveling could control the access of protons to the hemi-channels of subunit a during proton-pumping activity.…”

“…This swiveling could control the access of protons to the hemi-channels of subunit a during proton-pumping activity. (62) However, the NMR-based structure of a peptide, corresponding to TM7 helix, revealed that it is composed of two a-helical regions separated by a flexible hinge, which allow both helical segments to move independently to each other. (63) The authors suggested that this movement in TM7 cause the opening and closing of the proton hemi-channel and enables proton translocation.…”

“…In yeast, the N-terminal cytosolic domains of two a subunit isoforms (Vph1p and Stv1p) are responsible for the specific targeting of V-ATPase to vacuolar and Golgi compartments, respectively. (62,64) Remarkably, in Paramecium tetraurelia, the seventeenth isoform has been recently identified and C-terminal domain has been implicated in their specific targeting. (65) The a isoforms of mice and human (a1, a2, a3, a4) are localized in different endomembrane organelles and plasma membranes of specialized cells.…”

Conformational studies of peptides representing a segment of TM7 from H+-VO-ATPase in SDS micelles

“…1) (Duarte et al 2007a, b; Vos et al 2007) . These putative transmembrane peptides include the arginine residue (R735), which is known to be part of the proton translocation channel, interacting with the glutamic acids from the subunits c, c′, and c″ (Nishi and Forgac 2002).…”