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

Ternary interactions of E1G1, C1, and a1_NT.

<p>(A) Possible model of ternary binding interaction of E1G1, C1 and a1_NT. Dotted arrows indicate weak and solid arrows (black) strong binding. (B) Gel filtration profile of E1G1/C1/a1_NT mixture (red) in comparison to E1G1 (green), C1 (blue) and a1_NT (yellow) monomers. (C) SDS-PAGE analysis of the eluted fractions from gel filtration chromatography. Border colors indicate samples corresponding to the color scheme used in 5B. “C” indicates control proteins. (D) Panel X: Basic native polyacrylamide gel electrophoresis analysis of the E1G1/C1/a1_NT mixture. A 2∶1∶2 molar ratio of E1G1:C1:a1_NT proteins was prepared and incubated on ice for 1 h (lane 4). Bands corresponding to one molar amount of E1G1, C1 and a1_NT proteins are visible in lanes 1, 2, and 3, respectively. Panel Y: SDS-PAGE (12% gel) analysis of the strong band eluted from the native gel in panel X (lane 4), suggesting the presence of C1, E1, and G1 in the complex. An unbound a1_NT band was observed at the expected position. (E) Model showing the binding mode interpreted from the Biacore data (inset) where ligand was C1: X, the binding model of E1G1 as analyte (10 µM protein). Y, the binding model of a1_NT as analyte (10 µM protein). Z, the binding model of E1G1 and a1_NT as analytes (10 µM of each protein). The inset shows the sensorgrams of X, Y, and Z binding interactions (See details in the text).</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

Ternary interactions of E1G1, C1, and H.

<p>(A) Model of E1G1-C1-H assembly. Dotted arrows (red) and solid arrows (black) indicate weak and strong binding, respectively. (B) Gel filtration profile of the H/C1/E1G1 mixture (red) in comparison to H (purple), E1G1 (green), and C1 (blue) monomers. (C) SDS-PAGE analysis of the eluted fractions from gel filtration chromatography. Border colors indicate samples corresponding to the color scheme used in 4B. “C” indicates control proteins. (D) Panel X: Basic native polyacrylamide gel electrophoresis analysis of the H/C1/E1G1 mixture. A molar ratio of 3∶1∶1 of E1G1:C1:H proteins was prepared and incubated on ice for 1 h (lane 4). Bands corresponding to one molar amount of E1G1, C1, and H proteins are visible in lanes 1, 2, and 3, respectively. Panel Y: SDS-PAGE (12% gel) analysis of the E1G1-C1-H mixture band eluted from the native gel in panel X (lane 4). (E) SDS-PAGE of the eluted proteins from the His-tag pulldown experiment. Lane 1, fraction eluted using buffer B; lane 2, subunits bound with His-tagged H subunit eluted using buffer C.</p

Interactions between E1G1 and C1.

<p>(A) Mode of E1G1-C1 binding interaction <i>in vitro</i> based on data reported from previous studies of yeast <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0055704#pone.0055704-Oot1" target="_blank">[11]</a>. Using a Biacore system, the <i>K_D</i> values for affinities of C1_head-E1G1 and C1_foot-E1G1 were estimated to be 2.8 nM and 1.9 µM, respectively, as shown in 2D. (B) Gel filtration profile of E1G1/C1 complex formation (red) in comparison with E1G1 (green) and C1 (blue) 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 2B. “C” indicates control proteins. (D) Panel X: Basic native polyacrylamide gel electrophoresis analysis of E1G1 and C1 interaction. For complex formation, a 2∶1 molar ratio of E1G1:C1 proteins was prepared and incubated on ice for 1 h (lane 3). Bands corresponding to one molar amount of E1G1 and C1 are visible in lanes 1 and 2, respectively. Panel Y: SDS-PAGE (12% gel) analysis of the E1G1C1 complex band eluted from the native gel in panel X (lane 3). (E) Real-time binding evaluation was performed using a Biacore system. Sensorgrams for the binding of various concentrations of the analyte (E1G1) to the ligand (C1) are shown. The inset curve shows the steady-state binding isotherm for binding of E1G1 at various concentrations to C1 ligand on a CM5 sensor chip.</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

Basic native-PAGE patterns for the reconstitution of wild-type/mutant catalytic domains (V₁ domains).

<p>Purified wild-type or mutant A ₃B₃ and DF complexes were mixed in a 1: 5 molar ratio and incubated on ice for 1 h to reconstitute the catalytic domain A ₃B₃DF, as described in Materials and Methods. Lanes 1, 10, 12, and 14: purified wild-type and mutant A ₃B₃ complexes; lane 2: wild-type A ₃B₃DF; lanes 3, 9, 11, 13, 15, 17: reconstituted mutant catalytic domains; and lanes 18 and 19: B and A monomers, respectively. Three micrograms of proteins were loaded in lanes 1, 9, 16, and 17, and 2 µg in lanes 10, 15, 18, and 19.</p

Schematic model of <i>E</i><i>. hirae</i> V-ATPase (adapted from [15] and [16]).

<p>The V₁ domain of V-ATPase is composed of a hexameric arrangement of alternating A and B subunits responsible for ATP binding and hydrolysis; it also contains the DF subunits (shown by a dotted red line), the focus of this study. The V_o domain of V-ATPase comprises an a subunit and an attached membrane c ring. The V₁ and V_o domains are connected by a central stalk, which is composed of D, F, and d subunits, and 2 peripheral stalks assembled from the E and G subunits of V₁. ATP hydrolysis induces the rotation of the central axis (DFd complex) together with the c ring, which causes Na⁺ to be pumped through the channel at the interface between the c ring and the a subunit.</p

ATPase activities and their stability in mutant A₃B₃DF complexes of <i>E</i><i>. hirae</i> V-ATPase.

<p>ATPase activities of the mutants were measured using an ATP regeneration system as described in Materials and Methods. <i>A</i>, ATPase activities of the central-axis D subunit mutants measured using various concentrations of ATP. <i>B</i>, Lineweaver-Burk plots of the ATPase activities from <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0074291#pone-0074291-g004" target="_blank">Figure 4A</a> used for calculating <i>K</i>_m and <i>V</i>_max values for the D mutants. <i>C</i>-<i>D</i>, Stability of ATPase activities of mutant A ₃B₃DF complexes. ATPase activities were measured in the presence of 1 mM ATP. <i>Filled </i><i>circles</i>, A ₃B₃D(RR^165-6AA) F; <i>open </i><i>diamonds</i>, A ₃B₃D(R¹⁶⁶ A) F; <i>filled </i><i>diamonds</i>, A ₃B₃D(R¹⁶⁵ A) F; <i>filled </i><i>triangles</i>, A ₃B(V³⁸⁸ A) ₃D(RR^165-6AA) F; <i>open </i><i>triangles</i>, A(R⁴⁷⁵ A) ₃B₃D(RR^165-6AA) F; <i>filled </i><i>squares</i>, A ₃B(L³⁸⁹ A) ₃DF; <i>open </i><i>squares</i>, A(R⁴⁷⁵ A) ₃B₃DF; <i>open </i><i>crosses</i>, A ₃B(V³⁸⁸ A) ₃DF; and <i>open </i><i>circles</i>, wild-type A ₃B₃DF.</p