Binding interactions of the peripheral stalk subunit isoforms from human V-ATPase

Figures & data

Fig. 1. Schematic model of human V-ATPase illustrating the mode of binding interactions at the peripheral stalk region.

Notes: The approximate molecular weight of each peripheral stalk subunit along with the function and the number of isoforms are stated. Diverse affinities and kinetics from the binding interactions of the subunits/isoforms were observed from the real time surface plasmon resonance (SPR) quantitative system. The interaction of C1-E1G3 shown as an example.

Fig. 2. Interactions between EG isoforms and C2.

Notes: (A) Gel filtration profile of E1G1-C2 complex formation (red) compared with that of E1G1 (green) and C2 (blue), and SDS-PAGE analysis of the eluted fractions from gel filtration chromatography. Gel border colors indicate samples corresponding to the color scheme used in the chromatogram. (B) Panel X: Basic native PAGE analysis of E2G1 and C2 interaction. For complex formation, a 2:1 M ratio of E2G1:C2 proteins were prepared and incubated on ice for 1 h (lane 3). Panel Y: SDS-PAGE profile of the bands (A & B) eluted from the native gel in panel X (lane 3).

Table 1. Kinetic parameters of binary subunit–subunit binding interactions obtained by Biacore experiments.

Ligand (Molecular Mass)	Analyte	Association rate ka (M^−1s^−1)	Dissociation rate kd (s^−1)	Affinity KD (M)	Reference
C1 (44,460)	E1G1	3.7 × 10^6	0.011	2.8 × 10^−9	(27)
C1 (44,460)	E1G1	5.4 × 10^2	0.01	1.9 × 10^−6	(27)
C1 (44,460)	E1G2	8.1 × 10^5	0.00134	1.7 × 10^−9	This study
C1 (44,460)	E1G2	4770	0.0093	2.0 × 10^−6	This study
C1 (44,460)	E1G3	9.5 × 10^4	0.001306	1.4 × 10^−8	This study
C1 (44,460)	E1G3	3473	0.01063	3.1 × 10^−6	This study
H (56,402)	E1G1	–	–	4.8 × 10^−8	(27)
H (56,402)	E1G2	–	–	1.1 × 10^−7	This study
H (56,402)	E1G3	–	–	5.7 × 10^−8	This study
H (56,402)	C1	–	–	1.4 × 10^−6	(27)
H (56,402)	C2	–	–	1.7 × 10^−6	This study
C1 (44,460)	a1NT	–	–	3.0 × 10^−6	(27)
C1 (44,460)	a2NT	–	–	3.5 × 10^−6	This study
a1NT (41,532)	E1G1	–	–	2.1 × 10^−7	(27)
a1NT (41,532)	E1G2	–	–	2.0 × 10^−7	This study
a1NT (41,532)	E1G3	–	–	4.7 × 10^−8	This study
H (56,402)	a1NT	–	–	3.6 × 10^−7	(27)
H (56,402)	a2NT	–	–	2.1 × 10^−7	This study

Fig. 3. Interactions of EG-isoforms with the H subunit.

Notes: (A) and (B) Interactions of E1G2-H, and E1G3-H mixtures, respectively. Left, gel filtration profile of the E1G2-H and E1G3-H mixtures (red) in comparison to H (purple) and EG (green) monomers. Middle, SDS-PAGE analysis of the eluted fractions from gel filtration chromatography. Right, basic native PAGE analysis of E1G2-H and E1G3-H interactions (panel X). For complex formation, equimolar amounts of EG-isoforms (independently) and H proteins were mixed and incubated on ice for 1 h (lane 3). Panel Y: SDS-PAGE profile of the bands eluted from the native gel in panel X (lane 3).

Fig. 4. Binary and ternary interactions of H-C2-isoform.

Notes: (A) SDS-PAGE analysis of the pull-down assay of H-C2 interactions. Lane 1, fraction eluted using buffer B; lane 2, subunits bound with His-tagged H subunit eluted using buffer C. Negative control, C2 without His-tagged H subunit. (B) SDS-PAGE analysis of the pull-down assay of H-C2-E1G1 interaction. Lane 1, fraction eluted using buffer B; lane 2, subunits bound with His-tagged H subunit eluted using buffer C. (C) Panel X: Basic native PAGE analysis of H-C2-E1G1 interaction. A molar ratio of 3:1:1 of E1G1:C2:H proteins were prepared and incubated on ice for 1 h (lane 4). Bands corresponding to 1 molar amount of H, C2, and E1G1 proteins are visible in lanes 1, 2, and 3, respectively, on native gel. Panel Y: SDS-PAGE profile of the complex band and other bands (A, B, and C) eluted from the native gel in panel X (lane 4) of H-C2-E1G1 interactions, respectively.

Fig. 5. Quaternary binding interactions.

Notes: (A) and (B) Left, interactions E1G1-C2-a2-H, and E1G1-C1-a2-H mixtures, respectively. Gel filtration profile of the E1G1-C2-a2-H and E1G1-C1-a2-H mixture (red) in comparison to H (purple), E1G1 (green), C1/C2 (blue), and a2 (yellow) monomers. Middle, SDS-PAGE analysis of the eluted fractions from gel filtration chromatography. Right, SDS-PAGE analysis of the pull-down assay of E1G1-C2-a2-H, and E1G1-C1-a2-H interactions. Lane 1, fraction eluted using buffer B; lane 2, subunits bound with His-tagged H subunit eluted using buffer C.

Fig. 6. Multiple sequence alignment of yeast C (Vma5p), C1, and C2 subunit isoforms of human V-ATPase with other C1 and C2 subunit isoforms from different species. Conserved amino acids are highlighted by purple color. The amino acids constituting the head (red bar), neck (green bar), and foot (blue bar) regions are highlighted above the sequence according to the crystal structure of the yeast C subunit. Residues involved in dimer interfaces from PISA analysis are highlighted as yellow. Hs, H. sapiens; Mm, M. musculus; Xt, X. tropicalis; Dr, Danio rerio.

Fig. 7. Sequence alignment of the C1 and C2 subunit isoforms of human V-ATPase with other C1 and C2 subunit isoforms from different species.

Notes: Pink and blue boxes highlight conserved amino acids only among C1 and C2 isoforms from different species, respectively. Hs, H. sapiens; Mm, M. musculus; Xt, X. tropicalis; Dr, Danio rerio.

Fig. 8. Consurf conservation analysis for the homology models.

Notes: (A) Conservation among C1 isoforms on C1 model, (B) conservation among C1 and C2 isoforms on C1 model, (C) conservation among C1 and C2 isoforms on C2 model.