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

<p>The mammalian peripheral stalk subunits of the vacuolar-type H⁺-ATPases (V-ATPases) possess several isoforms (C1, C2, E1, E2, G1, G2, G3, a1, a2, a3, and a4), which may play significant role in regulating ATPase assembly and disassembly in different tissues. To better understand the structure and function of V-ATPase, we expressed and purified several isoforms of the human V-ATPase peripheral stalk: E1G1, E1G2, E1G3, E2G1, E2G2, E2G3, C1, C2, H, a1_NT, and a2_NT. Here, we investigated and characterized the isoforms of the peripheral stalk region of human V-ATPase with respect to their affinity and kinetics in different combination. We found that different isoforms interacted in a similar manner with the isoforms of other subunits. The differences in binding affinities among isoforms were minor from our <i>in vitro</i> studies. However, such minor differences from the binding interaction among isoforms might provide valuable information for the future structural-functional studies of this holoenzyme.</p> <p>Schematic model of human V-ATPase illustrating the mode of binding interactions at the peripheral stalk region.</p

Structural model of human V-ATPase.

<p>Peripheral stalk subunits (E, G, C, a_NT, and H) are emphasized using colors in the schematic model.</p

Interactions between E1G1 and H.

<p>(A) Possible mode of E1G1-H binding interaction <i>in vitro</i>. Using a Biacore system, the <i>K_D</i> values for affinity of H-E1G1 was estimated to be 48 nM. (B) Gel filtration profile of E1G1/H complex formation (red) in comparison to E1G1 (green) and H (purple) monomers. (C) SDS-PAGE analysis of the eluted fractions from gel filtration chromatography. Gel Border colors indicate samples corresponding to the color scheme used in 3B. “C” indicates control proteins. (D) Panel X: Basic native polyacrylamide gel electrophoresis analysis of E1G1 and H interaction. For complex formation, equimolar amounts of E1G1 and H proteins were mixed and incubated on ice for 1 h (lane 3). Bands corresponding to one molar amount of E1G1 and H are visible in lanes 1 and 2, respectively. Panel Y: SDS-PAGE (12% gel) analysis of the E1G1H complex band eluted from the native gel in panel X (lane 3). (E) SDS-PAGE of the eluted proteins from the His-tag pulldown experiment. Lane 1, fractions eluted using buffer B; lane 2, E1G1 complex bound with His-tagged H subunit eluted using buffer C. (F) Real-time binding evaluation was performed using a Biacore system. Sensorgrams for the binding of various concentrations of the analyte (E1G1) to the ligand (H) are shown. The inset curve shows the steady-state binding isotherm for binding of E1G1 at various concentrations to H ligand on a CM5 sensor chip.</p