[Show abstract][Hide abstract] ABSTRACT: Vacuolar ATPase (V-ATPase) of Enterococcus hirae is composed of a soluble functional domain V1 (A3B3DF) and an integral membrane domain Vo (ac), where V1 and Vo domains are connected by a central stalk, composed of D-, F-, and d-subunits; and two peripheral stalks (E- and G-subunits). We identified 120 interacting residues of A3B3 heterohexamer with D-subunit in DF heterodimer in the crystal structures of A3B3 and A3B3DF. In our previous study, we reported 10 mutants of E. hirae V1-ATPase, which showed lower binding affinities of DF with A3B3 complex leading to higher initial specific ATPase activities compared to the wild-type. In this study, we identified a mutation of A-subunit (LV(476-7)AA) at its C-terminal domain resulting in the A3B3 complex with higher binding affinities for wild-type or mutant DF heterodimers and lower initial ATPase activities compared to the wild-type A3B3 complex, consistent with our previous proposal of reciprocal relationship between the ATPase activity and the protein-protein binding affinity of DF axis to the A3B3 catalytic domain of E. hirae V-ATPase. These observations suggest that the binding of DF axis at the contact region of A3B3 rotary ring is relevant to its rotation activity.
[Show abstract][Hide abstract] ABSTRACT: Vacuolar ATPases (V-ATPases) function as proton pumps in various cellular membrane systems. The hydrophilic V1 portion of the V-ATPase is a rotary motor, in which a central-axis DF complex rotates inside a hexagonally arranged catalytic A3B3 complex by using ATP hydrolysis energy. We have previously reported crystal structures of Enterococcushirae V-ATPase A3B3 and A3B3DF (V1) complexes; the result suggested that the DF axis induces structural changes in the A3B3 complex through extensive protein-protein interactions. In this study, we mutated 10 residues at the interface between A3B3 and DF complexes and examined the ATPase activities of the mutated V1 complexes as well as the binding affinities between the mutated A3B3 and DF complexes. Surprisingly, several V1 mutants showed higher initial ATPase activities than wild-type V1-ATPase, whereas these mutated A3B3 and DF complexes showed decreased binding affinities for each other. However, the high ATP hydrolysis activities of the mutants decreased faster over time than the activity of the wild-type V1 complex, suggesting that the mutants were unstable in the reaction because the mutant A3B3 and DF complexes bound each other more weakly. These findings suggest that strong interaction between the DF complex and A3B3 complex lowers ATPase activity, but also that the tight binding is responsible for the stable ATPase activity of the complex.
PLoS ONE 09/2013; 8(9):e74291. DOI:10.1371/journal.pone.0074291 · 3.23 Impact Factor
[Show abstract][Hide abstract] 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.
PLoS ONE 02/2013; 8(2):e55704. DOI:10.1371/journal.pone.0055704 · 3.23 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: In various cellular membrane systems, vacuolar ATPases (V-ATPases) function as proton pumps, which are involved in many processes such as bone resorption and cancer metastasis, and these membrane proteins represent attractive drug targets for osteoporosis and cancer. The hydrophilic V(1) portion is known as a rotary motor, in which a central axis DF complex rotates inside a hexagonally arranged catalytic A(3)B(3) complex using ATP hydrolysis energy, but the molecular mechanism is not well defined owing to a lack of high-resolution structural information. We previously reported on the in vitro expression, purification and reconstitution of Enterococcus hirae V(1)-ATPase from the A(3)B(3) and DF complexes. Here we report the asymmetric structures of the nucleotide-free (2.8 Å) and nucleotide-bound (3.4 Å) A(3)B(3) complex that demonstrate conformational changes induced by nucleotide binding, suggesting a binding order in the right-handed rotational orientation in a cooperative manner. The crystal structures of the nucleotide-free (2.2 Å) and nucleotide-bound (2.7 Å) V(1)-ATPase are also reported. The more tightly packed nucleotide-binding site seems to be induced by DF binding, and ATP hydrolysis seems to be stimulated by the approach of a conserved arginine residue. To our knowledge, these asymmetric structures represent the first high-resolution view of the rotational mechanism of V(1)-ATPase.
[Show abstract][Hide abstract] ABSTRACT: Vacuolar ATPase (V-ATPase) of Enterococcus hirae is composed of a soluble catalytic domain (V₁; NtpA₃-B₃-D-G) and an integral membrane domain (V₀; NtpI-K₁₀) connected by a central and two peripheral stalks (NtpC, NtpD-G and NtpE-F). Recently nucleotide binding of catalytic NtpA monomer has been reported (Arai et al.). In the present study, we calculated the nucleotide binding affinity of NtpA by molecular dynamics (MD) simulation/free energy calculation using MM-GBSA approach based on homology modeled structure of NtpA monomer docked with ATP analogue, adenosine 5'-[β, γ-imido] triphosphate (AMP-PNP). The calculated binding free energies showed qualitatively good agreement with experimental data. The calculation was cross-validated further by the rigorous method, thermodynamic integration (TI) simulation. Finally, the interaction between NtpA and nucleotides at the atomic level was investigated by the analyses of components of free energy and the optimized model structures obtained from MD simulations, suggesting that electrostatic contribution is responsible for the difference in nucleotide binding to NtpA monomer. This is the first observation and suggestion to explain the difference of nucleotide binding properties in V-ATPase NtpA subunit, and our method can be a valuable primary step to predict nucleotide binding affinity to other subunits (NtpAB, NtpA₃B₃) and to explore subunit interactions and eventually may help to understand energy transduction mechanism of E. hirae V-ATPase.
[Show abstract][Hide abstract] ABSTRACT: The selective inhibition of an aminopeptidase from Aeromonas proteolytica (AAP), a dinuclear Zn(2+) hydrolase, by 8-quinolinol (8-hydroxyquinoline, 8-HQ) derivatives is reported. We previously reported on the preparation of 8-HQ-pendant cyclens as Zn(2+) fluorophores (cyclen is 1,4,7,10-tetraazacyclododecane), in which the nitrogen and phenolate of the 8-HQ units (as well as the four nitrogens of cyclen) bind to Zn(2+) in a bidentate manner to form very stable Zn(2+) complexes at neutral pH (K (d) = 8-50 fM at pH 7.4). On the basis of this finding, it was hypothesized that 8-HQ derivatives have the potential to function as specific inhibitors of Zn(2+) enzymes, especially dinuclear Zn(2+) hydrolases. Assays of 8-HQ derivatives as inhibitors were performed against commercially available dinuclear Zn(2+) enzymes such as AAP and alkaline phosphatase. 8-HQ and the 5-substituted 8-HQ derivatives were found to be competitive inhibitors of AAP with inhibition constants of 0.16-29 μM at pH 8.0. The nitrogen at the 1-position and the hydroxide at the 8-position of 8-HQ were found to be essential for the inhibition of AAP. Fluorescence titrations of these drugs with AAP and an X-ray crystal structure analysis of an AAP-8-HQ complex (1.3-Å resolution) confirmed that 8-HQ binds to AAP in the "Pyr-out" mode, in which the hydroxide anion of 8-HQ bridges two Zn(2+) ions (Zn1 and Zn2) in the active site of AAP and the nitrogen atom of 8-HQ coordinates to Zn1 (Protein Data Bank code 3VH9).
European Journal of Biochemistry 02/2012; 17(4):517-29. DOI:10.1007/s00775-012-0873-4 · 2.54 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: V-ATPases function as ATP-dependent ion pumps in various membrane systems of living organisms. ATP hydrolysis causes rotation of the central rotor complex, which is composed of the central axis D subunit and a membrane c ring that are connected by F and d subunits. Here we determined the crystal structure of the DF complex of the prokaryotic V-ATPase of Enterococcus hirae at 2.0-Å resolution. The structure of the D subunit comprised a long left-handed coiled coil with a unique short β-hairpin region that is effective in stimulating the ATPase activity of V(1)-ATPase by twofold. The F subunit is bound to the middle portion of the D subunit. The C-terminal helix of the F subunit, which was believed to function as a regulatory region by extending into the catalytic A(3)B(3) complex, contributes to tight binding to the D subunit by forming a three-helix bundle. Both D and F subunits are necessary to bind the d subunit that links to the c ring. From these findings, we modeled the entire rotor complex (DFdc ring) of V-ATPase.
Proceedings of the National Academy of Sciences 11/2011; 108(50):19955-60. DOI:10.1073/pnas.1108810108 · 9.67 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Biacore is widely used for studies on protein-protein interaction in which regeneration is one of the most important steps. Here we introduce the anionic detergent sodium lauroyl sarcosinate (sarkosyl), which works satisfactorily as a regeneration reagent. After regeneration by the mild detergent, the subsequent binding experiment was reproducible without any degradation of the ligand. This regeneration condition can be employed for diverse combinations of ligand-analyte binding interactions and optimized as required.
[Show abstract][Hide abstract] ABSTRACT: Asp187 and Gln190 were predicted as conserved and closely located at the Na(+) binding site in a topology and homology model structure of Na(+)/proline symporter (PutP) of Escherichia coli. The replacement of Asp187 with Ala or Leu did not affect proline transport activity; whereas, change to Gln abolished the active transport. The binding affinity for Na(+) or proline of these mutants was similar to that of wild-type (WT) PutP. This result indicates Asp187 to be responsible for active transport of proline without affecting the binding. Replacement of Gln190 with Ala, Asn, Asp, Leu and Glu had no effect on transport or binding, suggesting that it may not have a role in the transport. However, in the negative D187Q mutant, a second mutation, of Gln190 to Glu or Leu, restored 46 or 7% of the transport activity of WT, respectively, while mutation to Ala, Asn or Asp had no effect. Thus, side chain at position 190 has a crucial role in suppressing the functional defect of the D187Q mutant. We conclude that Asp187 is responsible for transport activity instead of coupling-ion binding by constituting the translocation pathway of the ion and Gln190 provides a suppressing mutation site to regain PutP functional activity.
Journal of Biochemistry 05/2011; 150(4):395-402. DOI:10.1093/jb/mvr062 · 2.58 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: AMP-activated protein kinase (AMPK) is a serine/threonine kinase that functions as a sensor to maintain energy balance at both the cellular and the whole-body levels and is therefore a potential target for drug design against metabolic syndrome, obesity and type 2 diabetes. Here, the crystal structure of the phosphorylated-state mimic T172D mutant kinase domain from the human AMPK α2 subunit is reported in the apo form and in complex with a selective inhibitor, compound C. The AMPK α2 kinase domain exhibits a typical bilobal kinase fold and exists as a monomer in the crystal. Like the wild-type apo form, the T172D mutant apo form adopts the autoinhibited structure of the `DFG-out' conformation, with the Phe residue of the DFG motif anchored within the putative ATP-binding pocket. Compound C binding dramatically alters the conformation of the activation loop, which adopts an intermediate conformation between DFG-out and DFG-in. This induced fit forms a compound-C binding pocket composed of the N-lobe, the C-lobe and the hinge of the kinase domain. The pocket partially overlaps with the putative ATP-binding pocket. These three-dimensional structures will be useful to guide drug discovery.
[Show abstract][Hide abstract] ABSTRACT: The vacuolar-type H+-ATPase (V-ATPase) is a multi-subunit proton pump that is involved in both intra- and extracellular acidification processes throughout human body. Subunits constituting the peripheral stalk of the V-ATPase are known to have several isoforms responsible for tissue/cell specific different physiological roles. To study the different interaction of these isoforms, we expressed and purified the isoforms of human V-ATPase peripheral stalk subunits using Escherichia coli cell-free protein synthesis system: E1, E2, G1, G2, G3, C1, C2, H and N-terminal soluble part of a1 and a2 isoforms. The purification conditions were different depending on the isoforms, maybe reflecting the isoform specific biochemical characteristics. The purified proteins are expected to facilitate further experiments to study about the cell specific interaction and regulation and thus provide insight into physiological meaning of the existence of several isoforms of each subunit in V-ATPase.
Protein Expression and Purification 02/2011; 78(2):181-8. DOI:10.1016/j.pep.2011.02.012 · 1.70 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Enterococcus hirae vacuolar ATPase (V-ATPase) is composed of a soluble catalytic domain (V(1); NtpA(3)-B(3)-D-G) and an integral membrane domain (V(0); NtpI-K(10)) connected by a central and peripheral stalk(s) (NtpC and NtpE-F). Here we examined the nucleotide binding of NtpA monomer, NtpB monomer or NtpD-G heterodimer purified by using Escherichia coli expression system in vivo or in vitro, and the reconstitution of the V(1) portion with these polypeptides. The affinity of nucleotide binding to NtpA was 6.6 microM for ADP or 3.1 microM for ATP, while NtpB or NtpD-G did not show any binding. The NtpA and NtpB monomers assembled into NtpA(3)-B(3) heterohexamer in nucleotide binding-dependent manner. NtpD-G bound NtpA(3)-B(3) forming V(1) (NtpA(3)-B(3)-D-G) complex independent of nucleotides. The V(1) formation from individual NtpA and NtpB monomers with NtpD-G heterodimer was absolutely dependent on nucleotides. The ATPase activity of reconstituted V(1) complex was as high as that of native V(1)-ATPase purified from the V(0)V(1) complex by EDTA treatment of cell membrane. This in vitro reconstitution system of E. hirae V(1) complex will be valuable for characterizing the subunit-subunit interactions and assembly mechanism of the V(1)-ATPase complex.
Biochemical and Biophysical Research Communications 10/2009; 390(3):698-702. DOI:10.1016/j.bbrc.2009.10.032 · 2.30 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The vacuolar ATPase (V-ATPase) is composed of a soluble catalytic domain and an integral membrane domain connected by a central stalk and a few peripheral stalks. The number and arrangement of the peripheral stalk subunits remain controversial. The peripheral stalk of Na+-translocating V-ATPase from Enterococcus hirae is likely to be composed of NtpE and NtpF (corresponding to subunit G of eukaryotic V-ATPase) subunits together with the N-terminal hydrophilic domain of NtpI (corresponding to subunit a of eukaryotic V-ATPase). Here we purified NtpE, NtpF, and the N-terminal hydrophilic domain of NtpI (NtpI(Nterm)) as separate recombinant His-tagged proteins and examined interactions between these three subunits by pulldown assay using one tagged subunit, CD spectroscopy, surface plasmon resonance, and analytical ultracentrifugation. NtpI(Nterm) directly bound NtpF, but not NtpE. NtpE bound NtpF tightly. NtpI(Nterm) bound the NtpE-F complex stronger than NtpF only, suggesting that NtpE increases the binding affinity between NtpI(Nterm) and NtpF. Purified NtpE-F-I(Nterm) complex appeared to be monodisperse, and the molecular masses estimated from analytical ultracentrifugation and small-angle x-ray scattering (SAXS) indicated that the ternary complex is formed with a 1:1:1 stoichiometry. A low resolution structure model of the complex produced from the SAXS data showed an elongated "L" shape.
[Show abstract][Hide abstract] ABSTRACT: Calcium signals mediate a multitude of plant responses to external stimuli. Calcineurin B-like (CBL) proteins and their target kinases, CBL-interacting protein kinases (CIPKs), represent important relays in plant calcium signaling. CBL interacts with CIPK through a conserved motif (NAF/FISL motif) within the C-terminal regulatory domain. To better understand the functional role of the CBL-CIPK system, we determined the crystal structure of AtCBL2 in complex with the regulatory domain of AtCIPK14 at 1.2 A resolution. The NAF/FISL motif is inserted into a hydrophobic crevice within AtCBL2, accompanied by a large displacement of the helices and loop on the opposite side of the NAF/FISL motif from the C-terminal region, which shields the hydrophobic crevice in free form. Ca(2+) are coordinated within four EF hands in AtCBL2 in bound form. This calcium coordination pattern differs from that in the structure of the SOS3-SOS2 complex previously reported. Structural comparison of the two structures shows that the recognition of CBL by CIPK is performed in a similar manner, but inherent interactions confer binding affinity and specificity.