Crystal structure of the cytoplasmic N-terminal domain of subunit I, a homolog of subunit a, of V-ATPase

Abstract

Subunit "a" is associated with the membrane-bound (V(O)) complex of eukaryotic vacuolar H(+)-ATPase acidification machinery. It has also been shown recently to be involved in diverse membrane fusion/secretory functions independent of acidification. Here, we report the crystal structure of the N-terminal cytosolic domain from the Meiothermus ruber subunit "I" homolog of subunit a. The structure is composed of a curved long central α-helix bundle capped on both ends by two lobes with similar α/β architecture. Based on the structure, a reasonable model of its eukaryotic subunit a counterpart was obtained. The crystal structure and model fit well into reconstructions from electron microscopy of prokaryotic and eukaryotic vacuolar H(+)-ATPases, respectively, clarifying their orientations and interactions and revealing features that could enable subunit a to play a role in membrane fusion/secretion.

Fig. 1

X-ray and modeled structures in similar orientations. (a) Stereoview of the ribbon backbone trace of the I_cyt crystal structure color ramped from the N- to the C-terminal ends, and α-helices and β-sheet strands identified with Roman and Arabic numbers, respectively. Superposition of the distal (top) and proximal (bottom) lobes showed an rmsd of 5.3 Å over 23 α-carbons. (b) Space-filling representation of I_cyt structure. The long linker region of anti-parallel pairs of α-helices is colored green, and the distal and proximal lobes are distinguished by salmon and cyan colors, respectively. These distinctions are adopted in c to e. (c) Model of a_cyt of the neuronal zebrafish subunit a1 obtained as described in the Supplementary Methods and Table S3. The α-helices boxed in blue (αV) and red (αX) in the sequence alignment which are equivalent to I_cyt’s αIV and αX, respectively, have been implicated as sites for binding of t-SNARE protein syntaxin or SNAP25 and Ca²⁺-calmodulin, correspondingly (described further in Supplementary Fig. S3). (d) Ribbon trace and (e) space-filling representation, of the yeast subunit C (Vma5p) crystal structure (PDB 1U7L). The match from the DALI database between I_cyt and subunit C structure gave Z score of 6.8 and rmsd of 10.1 Å over 216 aligned residues and sequence identity of 7%. A large substructure is formed by extensive hydrophobic interactions of the C-terminal segment of the longest α helix (light green) of the linker with the inner face of β-sheet of the foot domain (blue).

Fig. 2

Fitting of the I_cyt crystal structure and a_cyt model to respective EM densities. The methods are described in the Supplementary Information. All the fits are unique as assessed by e3fhstat.py in EMAN2 which indicated 99^th percentile for volume inclusion and real-space correlation (for additional statistics see Supplementary Methods). (a) Fit of the crystal structures of I_cyt (color ramped ribbon trace) and the T. thermophilus heterodimer of subunits EG (gray trace) into the 16 Å cryo-EM density of the T. thermophilus V-ATPase. The surface density (available from ref. 15) of subunits A is colored yellow; B, red; D, blue; C, cyan; and c, magenta. The green and red arrows indicate the proximal and distal lobes of I_cyt, respectively. (b) Fit of the zebrafish a_cyt model (Fig. 1c) color ramped trace and crystal structures of yeast subunits H²⁰ and C¹⁴ into the 25 Å negative-stain EM reconstruction density of yeast V-ATPase. The black arrows in this figure and that in panel c indicate the vertical column of density of two of the three peripheral stalks. (c) Fit of the identical components identified in b into the 17 Å cryo-EM reconstruction density of M. sexta V-ATPase. Red dashed rectangle delineates an area of the V-ATPase fit that has been magnified in (d). In both Figs. b and c, the third peripheral stalk is in the “rear” and obstructed from view. Compared to yeast V-ATPase, in M. sexta, a_cyt and subunit H are rotated clockwise by ~25° and ~30°, respectively, about a horizontal axis, whereas subunit C is rotated clockwise by ~ 10° about an axis normal to the plane of the figure. This level of difference may be attributed to the differences in the imaging methods and the resulting resolutions. (d) A magnified region of the zebrafish a_cyt fit delineated in panel c. Potential binding sites for SNARE and Ca²⁺-calmodulin (CaM) are indicated by green and orange arrows, respectively.