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Biochemical and biophysical properties of interactions between subunits of the peripheral stalk region of human V-ATPase.

Rahman S, Yamato I, Saijo S, Mizutani K, Ishizuka-Katsura Y, Ohsawa N, Terada T, Shirouzu M, Yokoyama S, Iwata S, Murata T - PLoS ONE (2013)

Bottom Line: The putative ternary complex of C1-H-E1G1 was not much strong on co-incubation of these subunits, indicating that the two strong complexes of C1-E1G1 and H-E1G1 in cooperation with many other weak interactions may be sufficiently strong enough to withstand the torque of rotation during catalysis.We observed a partially stable quaternary complex (consisting of E1G1, C1, a1(NT), and H subunits) resulting from discrete peripheral subunit interactions stabilizing the complex through their intrinsic affinities.No binding was observed in the absence of E1G1 (using only H, C1, and a1(NT)); therefore, it is likely that, in vivo, the E1G1 heterodimer has a significant role in the initiation of subunit assembly.

View Article: PubMed Central - PubMed

Affiliation: Department of Biological Science and Technology, Tokyo University of Science, Chiba, Japan. srahman@rs.tus.ac.jp

ABSTRACT
Peripheral stalk subunits of eukaryotic or mammalian vacuolar ATPases (V-ATPases) play key roles in regulating its assembly and disassembly. In a previous study, we purified several subunits and their isoforms of the peripheral stalk region of Homo sapiens (human) V-ATPase; such as C1, E1G1, H, and the N-terminal cytoplasmic region of V(o), a1. Here, we investigated the in vitro binding interactions of the subunits at the stalk region and measured their specific affinities. Surface plasmon resonance experiments revealed that the subunit C1 binds the E1G1 heterodimer with both high and low affinities (2.8 nM and 1.9 µM, respectively). In addition, an E1G1-H complex can be formed with high affinity (48 nM), whereas affinities of other subunit pairs appeared to be low (∼0.21-3.0 µM). The putative ternary complex of C1-H-E1G1 was not much strong on co-incubation of these subunits, indicating that the two strong complexes of C1-E1G1 and H-E1G1 in cooperation with many other weak interactions may be sufficiently strong enough to withstand the torque of rotation during catalysis. We observed a partially stable quaternary complex (consisting of E1G1, C1, a1(NT), and H subunits) resulting from discrete peripheral subunit interactions stabilizing the complex through their intrinsic affinities. No binding was observed in the absence of E1G1 (using only H, C1, and a1(NT)); therefore, it is likely that, in vivo, the E1G1 heterodimer has a significant role in the initiation of subunit assembly. Multiple interactions of variable affinity in the stalk region may be important to the mechanism of reversible dissociation of the intact V-ATPase.

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Interactions between E1G1 and C1.(A) Mode of E1G1-C1 binding interaction in vitro based on data reported from previous studies of yeast [11]. Using a Biacore system, the KD values for affinities of C1head-E1G1 and C1foot-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.
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pone-0055704-g002: Interactions between E1G1 and C1.(A) Mode of E1G1-C1 binding interaction in vitro based on data reported from previous studies of yeast [11]. Using a Biacore system, the KD values for affinities of C1head-E1G1 and C1foot-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.

Mentions: Subunit-subunit interactions were examined by several methods to determine genuine binding between E1G1 (a representative of all EG subunits) and both C1 (a representative of C subunits) and H subunits. Previous studies in yeast have shown that the C subunit can interact with two EG subunit pairs with different affinities [8], [11] (Figure 2A). By mixing E1G1 and C1 in a 2∶1 molar ratio, the E1G1C1 complex eluted as a peak with a small subsequent shoulder earlier than the corresponding monomers (E1G1 complex or C1 monomer) from the gel filtration column (Figure 2B); since E1 and G1 alone were unstable, they were co-expressed and co-purified as E1G1 complex [22]. The eluted fractions were analyzed by SDS-PAGE, and the complex formation was confirmed by the appearance of the C1 and E1G1 complex bands in fractions eluted earlier than their monomers (Figure 2C). The shoulder peak contained small amount of unbound C1 and excess E1G1, which was visible in the later fractions as analyzed by SDS-PAGE. Densitometric analysis of SDS-PAGE gels was performed using the ImageJ software to compare both equimolar E1G1-C1 (data not shown) and a 2∶1 molar ratio of E1G1:C1. Using the staining intensity of C1, E1, and G1 bands (Figure 2C) and estimating protein amounts using standard curves determined by us (Figure S1), the binding stoichiometry of C1 vs. E1G1 was found to be 1∶1, suggesting that the low-affinity binding site of the C1-foot (C1foot) region [11] failed to interact with E1G1. The molecular size of the complex estimated (on the basis of the calibration curve of standard proteins) from the eluted position was approximately 220 kDa, which is much higher than the calculated molecular weight (41+44 = 85 kDa). There are two possible explanations for this early elution: Either dimer formation of the C1-E1G1 complex (85×2 = 170 kDa) or the L-shaped structure of this complex [10], [25]. We did not have any other strong evidence for the dimerization of this complex; hence, the inconsistency probably was due to the effect of the L-shape of the complex, which migrates in a different manner from globular-shaped proteins.


Biochemical and biophysical properties of interactions between subunits of the peripheral stalk region of human V-ATPase.

Rahman S, Yamato I, Saijo S, Mizutani K, Ishizuka-Katsura Y, Ohsawa N, Terada T, Shirouzu M, Yokoyama S, Iwata S, Murata T - PLoS ONE (2013)

Interactions between E1G1 and C1.(A) Mode of E1G1-C1 binding interaction in vitro based on data reported from previous studies of yeast [11]. Using a Biacore system, the KD values for affinities of C1head-E1G1 and C1foot-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.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3569449&req=5

pone-0055704-g002: Interactions between E1G1 and C1.(A) Mode of E1G1-C1 binding interaction in vitro based on data reported from previous studies of yeast [11]. Using a Biacore system, the KD values for affinities of C1head-E1G1 and C1foot-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.
Mentions: Subunit-subunit interactions were examined by several methods to determine genuine binding between E1G1 (a representative of all EG subunits) and both C1 (a representative of C subunits) and H subunits. Previous studies in yeast have shown that the C subunit can interact with two EG subunit pairs with different affinities [8], [11] (Figure 2A). By mixing E1G1 and C1 in a 2∶1 molar ratio, the E1G1C1 complex eluted as a peak with a small subsequent shoulder earlier than the corresponding monomers (E1G1 complex or C1 monomer) from the gel filtration column (Figure 2B); since E1 and G1 alone were unstable, they were co-expressed and co-purified as E1G1 complex [22]. The eluted fractions were analyzed by SDS-PAGE, and the complex formation was confirmed by the appearance of the C1 and E1G1 complex bands in fractions eluted earlier than their monomers (Figure 2C). The shoulder peak contained small amount of unbound C1 and excess E1G1, which was visible in the later fractions as analyzed by SDS-PAGE. Densitometric analysis of SDS-PAGE gels was performed using the ImageJ software to compare both equimolar E1G1-C1 (data not shown) and a 2∶1 molar ratio of E1G1:C1. Using the staining intensity of C1, E1, and G1 bands (Figure 2C) and estimating protein amounts using standard curves determined by us (Figure S1), the binding stoichiometry of C1 vs. E1G1 was found to be 1∶1, suggesting that the low-affinity binding site of the C1-foot (C1foot) region [11] failed to interact with E1G1. The molecular size of the complex estimated (on the basis of the calibration curve of standard proteins) from the eluted position was approximately 220 kDa, which is much higher than the calculated molecular weight (41+44 = 85 kDa). There are two possible explanations for this early elution: Either dimer formation of the C1-E1G1 complex (85×2 = 170 kDa) or the L-shaped structure of this complex [10], [25]. We did not have any other strong evidence for the dimerization of this complex; hence, the inconsistency probably was due to the effect of the L-shape of the complex, which migrates in a different manner from globular-shaped proteins.

Bottom Line: The putative ternary complex of C1-H-E1G1 was not much strong on co-incubation of these subunits, indicating that the two strong complexes of C1-E1G1 and H-E1G1 in cooperation with many other weak interactions may be sufficiently strong enough to withstand the torque of rotation during catalysis.We observed a partially stable quaternary complex (consisting of E1G1, C1, a1(NT), and H subunits) resulting from discrete peripheral subunit interactions stabilizing the complex through their intrinsic affinities.No binding was observed in the absence of E1G1 (using only H, C1, and a1(NT)); therefore, it is likely that, in vivo, the E1G1 heterodimer has a significant role in the initiation of subunit assembly.

View Article: PubMed Central - PubMed

Affiliation: Department of Biological Science and Technology, Tokyo University of Science, Chiba, Japan. srahman@rs.tus.ac.jp

ABSTRACT
Peripheral stalk subunits of eukaryotic or mammalian vacuolar ATPases (V-ATPases) play key roles in regulating its assembly and disassembly. In a previous study, we purified several subunits and their isoforms of the peripheral stalk region of Homo sapiens (human) V-ATPase; such as C1, E1G1, H, and the N-terminal cytoplasmic region of V(o), a1. Here, we investigated the in vitro binding interactions of the subunits at the stalk region and measured their specific affinities. Surface plasmon resonance experiments revealed that the subunit C1 binds the E1G1 heterodimer with both high and low affinities (2.8 nM and 1.9 µM, respectively). In addition, an E1G1-H complex can be formed with high affinity (48 nM), whereas affinities of other subunit pairs appeared to be low (∼0.21-3.0 µM). The putative ternary complex of C1-H-E1G1 was not much strong on co-incubation of these subunits, indicating that the two strong complexes of C1-E1G1 and H-E1G1 in cooperation with many other weak interactions may be sufficiently strong enough to withstand the torque of rotation during catalysis. We observed a partially stable quaternary complex (consisting of E1G1, C1, a1(NT), and H subunits) resulting from discrete peripheral subunit interactions stabilizing the complex through their intrinsic affinities. No binding was observed in the absence of E1G1 (using only H, C1, and a1(NT)); therefore, it is likely that, in vivo, the E1G1 heterodimer has a significant role in the initiation of subunit assembly. Multiple interactions of variable affinity in the stalk region may be important to the mechanism of reversible dissociation of the intact V-ATPase.

Show MeSH
Related in: MedlinePlus