Assembly of subunit d (Vma6p) and G (Vma10p) and the NMR solution structure of subunit G (G1-59) of the Saccharomyces cerevisiae V1VO ATPase

Understanding the structural traits of subunit G is essential, as it is needed for V₁V_O assembly and function. Here solution NMR of the recombinant N- (G_1-59) and C-terminal segment (G_61-114) of subunit G, has been performed in the absence and presence of subunit d of the yeast V-ATPase. The data show that G does bind to subunit d via its N-terminal part, G_1-59 only. The residues of G_1-59 involved in d binding are Gly7 to Lys34. The structure of G_1-59 has been solved, revealing an α-helix between residues 10 and 56, whereby the first nine- and the last three residues of G_1-59 are flexible. The surface charge distribution of G_1-59 reveals an amphiphilic character at the N-terminus due to positive and negative charge distribution at one side and a hydrophobic surface on the opposite side of the structure. The C-terminus exhibits a strip of negative residues. The data imply that G_1-59-d assembly is accomplished by hydrophobic interactions and salt-bridges of the polar residues. Based on the recently determined NMR structure of segment E_18-38 of subunit E of yeast V-ATPase and the presently solved structure of G_1-59, both proteins have been docked and binding epitopes have been analyzed.     

Spectroscopical identification of residues of subunit G of the yeast V-ATPase in its connection with subunit E

A critical point in the V₁ sector and entire V₁V_O complex is the interaction of stalk subunits G (Vma10p) and E (Vma4p). Previous work, using precipitation assays, has shown that both subunits form a complex. In this work, we have analysed the N-terminal segment of subunit G (G_1–59) of the V₁V_O ATPase from Saccharomyces cerevisiae by using nuclear magnetic resonance (NMR) spectroscopy. Analyses of ¹H-¹⁵N heteronuclear single quantum coherence (HSQC) spectra of G_1–59 in the absence and presence of the N-terminal peptides E_1–18 and E_18–38 as well as the produced and purified C-terminal segment (E_39–233) shows specific interactions only with the peptide fragment E_18–38. The binding of this peptide occurs via the residues M₁, V₂, S₃, and K₅ as well for V₂₂, S₂₃, K₂₄, A₂₅ and R₂₆ of G_1–59. The specific E_18–38/G_1–59 binding has been confirmed by fluorescence correlation spectroscopy data. The E_18–38 peptide has been studied by CD spectroscopy and NMR. The 3D structure of this peptide adopts a stable helix-hinge-helix formation in solution. A model structure of the E_18–38/G_1–59 complex reveals the orientation of E_18–38 relative to G_1–59 via salt-bridges of the polar residues and van der Waals forces at the very N-terminus of both segments.     

The boxing glove shape of subunit <Emphasis Type="Italic">d</Emphasis> of the yeast V-ATPase in solution and the importance of disulfide formation for folding of this protein

The low resolution structure of subunit d (Vma6p) of the Saccharomyces cerevisiae V-ATPase was determined from solution X-ray scattering data. The protein is a boxing glove-shaped molecule consisting of two distinct domains, with a width of about 6.5 nm and 3.5 nm, respectively. To understand the importance of the N- and C-termini inside the protein, four truncated forms of subunit d (d _11–345, d _38–345, d _1–328 and d _1–298) and mutant subunit d, with a substitution of Cys329 against Ser, were expressed, and only d _11–345, containing all six cysteine residues was soluble. The structural properties of d depends strongly on the presence of a disulfide bond. Changes in response to disulfide formation have been studied by fluorescence- and CD spectroscopy, and biochemical approaches. Cysteins, involved in disulfide bridges, were analyzed by MALDI-TOF mass spectrometry. Finally, the solution structure of subunit d will be discussed in terms of the topological arrangement of the V₁V_O ATPase.     

Interaction between subunit C (Vma5p) of the yeast vacuolar ATPase and the stalk of the C-depleted V1 ATPase from Manduca sexta midgut

Projection maps of a V₁-Vma5p hybrid complex, composed of subunit C (Vma5p) of Saccharomyces cerevisiae V-ATPase and the C-depleted V₁ from Manduca sexta, were determined from single particle electron microscopy. V₁-Vma5p consists of a headpiece and an elongated wedgelike stalk with a 2.1×3.0 nm protuberance and a 9.5×7.5 globular domain, interpreted to include Vma5p. The interaction face of Vma5p in V₁ was explored by chemical modification experiments.     

pH dependent inactivation of solubilized F1F0 ATP synthase by dicyclohexylcarbodiimide: pKa of detergent unmasked aspartyl-61 in Escherichia coli subunit c

The pH dependence of the reaction of dicyclohexylcarbodiimide with the essential aspartyl-61 residue in subunit c of Escherichia coli ATP synthase was compared in membranes and in a detergent dispersed preparation of the enzyme. The rate of reaction was estimated by measuring the inactivation of ATPase activity. The reaction with the detergent dispersed form of the enzyme proved to be pH sensitive with the essential aspartyl group titrating with a pK_a=8. However, when measured with E. coli membranes, the reaction proved to be pH insensitive. The results suggest that the reacting aspartyl-61 residues are shielded from the bulk aqueous solvent when in the membrane, but then become aqueous-accessible following detergent solubilization.     

Structural Analysis of the N-terminal Domain of Subunit a of the Yeast Vacuolar ATPase (V-ATPase) Using Accessibility of Single Cysteine Substitutions to Chemical Modification

The vacuolar ATPase (V-ATPase) is a multisubunit complex that carries out ATP-driven proton transport. It is composed of a peripheral V₁ domain that hydrolyzes ATP and an integral V₀ domain that translocates protons. Subunit a is a 100-kDa integral membrane protein (part of V₀) that possesses an N-terminal cytoplasmic domain and a C-terminal hydrophobic domain. Although the C-terminal domain functions in proton transport, the N-terminal domain is critical for intracellular targeting and regulation of V-ATPase assembly. Despite its importance, there is currently no high resolution structure for subunit a of the V-ATPase. Recently, the crystal structure of the N-terminal domain of the related subunit I from the archaebacterium Meiothermus ruber was reported. We have used homology modeling to construct a model of the N-terminal domain of Vph1p, one of two isoforms of subunit a expressed in yeast. To test this model, unique cysteine residues were introduced into a Cys-less form of Vph1p and their accessibility to modification by the sulfhydryl reagent 3-(N-maleimido-propionyl) biocytin (MPB) was determined. In addition, accessibility of introduced cysteine residues to MPB modification was compared in the V₁V₀ complex and the free V₀ domain to identify residues protected from modification by the presence of V₁. The results provide an experimental test of the proposed model and have identified regions of the N-terminal domain of subunit a that likely serve as interfacial contact sites with the peripheral V₁ domain. The possible significance of these results for in vivo regulation of V-ATPase assembly is discussed.     

Structural and functional analysis of the coupling subunit F in solution and topological arrangement of the stalk domains of the methanogenic A1AO ATP synthase

The first low-resolution shape of subunit F of the A₁A_O ATP synthase from the archaeon Methanosarcina mazei Gö1 in solution was determined by small angle X-ray scattering. Independent to the concentration used, the protein is monomeric and has an elongated shape, divided in a main globular part with a length of about 4.5 nm, and a hook-like domain of about 3.0 nm in length. The subunit-subunit interaction of subunit F inside the A₁A_O ATP synthase in the presence of 1-ethyl-3-(dimethylaminopropyl)-carbodiimide EDC was studied as a function of nucleotide binding, demonstrating movements of subunits F relative to the nucleotide-binding subunit B. Furthermore, in the intact A₁A_O complex, crosslinking of subunits D-E, A-H and A-B-D was obtained and the peptides, involved, were analyzed by MALDI-TOF mass spectrometry. Based on these data the surface of contact of B-F could be mapped in the high-resolution structure of subunit B of the A₁A_O ATP synthase.     

Molecular Basis of ADP Inhibition of Vacuolar (V)-type ATPase/Synthase

Reduction of ATP hydrolysis activity of vacuolar-type ATPase/synthase (V₀V₁) as a result of ADP inhibition occurs as part of the normal mechanism of V₀V₁ of Thermus thermophilus but not V₀V₁ of Enterococcus hirae or eukaryotes. To investigate the molecular basis for this difference, domain-swapped chimeric V₁ consisting of both T. thermophilus and E. hirae enzymes were generated, and their function was analyzed. The data showed that the interaction between the nucleotide binding and C-terminal domains of the catalytic A subunit from E. hirae V₁ is central to increasing binding affinity of the chimeric V₁ for phosphate, resulting in reduction of the ADP inhibition. These findings together with a comparison of the crystal structures of T. thermophilus V₁ with E. hirae V₁ strongly suggest that the A subunit adopts a conformation in T. thermophilus V₁ different from that in E. hirae V₁. This key difference results in ADP inhibition of T. thermophilus V₁ by abolishing the binding affinity for phosphate during ATP hydrolysis.     

Association of the eukaryotic V1VO ATPase subunits a with d and d with A

Owing to the complex nature of V₁V_O ATPases, identification of neighboring subunits is essential for mechanistic understanding of this enzyme. Here, we describe the links between the V₁ headpiece and the V_O-domain of the yeast V₁V_O ATPase via subunit A and d as well as the V_O subunits a and d using surface plasmon resonance and fluorescence correlation spectroscopy. Binding constants of about 60 and 200 nM have been determined for the a-d and d-A assembly, respectively. The data are discussed in light of subunit a and d forming a peripheral stalk, connecting the catalytic A₃B₃ hexamer with V_O.

Structured summary

MINT-7012054: d (uniprotkb:P32366) binds (MI:0407) to A (uniprotkb:P17255) by fluorescence correlation spectroscopy (MI:0052)MINT-7012041: d (uniprotkb:P32366) binds (MI:0407) to A (uniprotkb:P17255) by surface plasmon resonance (MI:0107)MINT-7012028: d (uniprotkb:P32366) binds (MI:0407) to a (uniprotkb:P32563) by surface plasmon resonance (MI:0107)     

The vacuolar (H+)-ATPase: subunit arrangement and in vivo regulation

The V-ATPases are responsible for acidification of intracellular compartments and proton transport across the plasma membrane. They play an important role in both normal processes, such as membrane traffic, protein degradation, urinary acidification, and bone resorption, as well as various disease processes, such as viral infection, toxin killing, osteoporosis, and tumor metastasis. V-ATPases contain a peripheral domain (V₁) that carries out ATP hydrolysis and an integral domain (V₀) responsible for proton transport. V-ATPases operate by a rotary mechanism involving both a central rotary stalk and a peripheral stalk that serves as a stator. Cysteine-mediated cross-linking has been used to localize subunits within the V-ATPase complex and to investigate the helical interactions between subunits within the integral V₀ domain. An essential property of the V-ATPases is the ability to regulate their activity in vivo. An important mechanism of regulating V-ATPase activity is reversible dissociation of the complex into its component V₁ and V₀ domains. The dependence of reversible dissociation on subunit isoforms and cellular environment has been investigated. Qi and Wang contributed equally to this work.     

Domain Features of the Peripheral Stalk Subunit H of the Methanogenic A1AO ATP Synthase and the NMR Solution Structure of H1-47

A series of truncated forms of subunit H were generated to establish the domain features of that protein. Circular dichroism analysis demonstrated that H is divided at least into a C-terminal coiled-coil domain within residues 54-104, and an N-terminal domain formed by adjacent α-helices. With a cysteine at the C-terminus of each of the truncated proteins (H_1-47, H_1-54, H_1-59, H_1-61, H_1-67, H_1-69, H_1-71, H_1-78, H_1-80, H_1-91, and H_47-105), the residues involved in formation of the coiled-coil interface were determined. Proteins H_1-54, H_1-61, H_1-69, and H_1-80 showed strong cross-link formation, which was weaker in H_1-47, H_1-59, H_1-71, and H_1-91. A shift in disulfide formation between cysteins at positions 71 and 80 reflected an interruption in the periodicity of hydrophobic residues in the region ₇₁AEKILEETEKE₈₁. To understand how the N-terminal domain of H is formed, we determined for the first time, to our knowledge, the solution NMR structure of H_1-47, which revealed an α-helix between residues 15-42 and a flexible N-terminal stretch. The α-helix includes a kink that would bring the two helices of the C-terminus into the coiled-coil arrangement. H_1-47 revealed a strip of alanines involved in dimerization, which were tested by exchange to single cysteines in subunit H mutants.     

Effects of mutations in the beta subunit hinge domain on ATP synthase F1 sector rotation: interaction between Ser 174 and Ile 163

A complex of γ, ε, and c subunits rotates in ATP synthase (F_oF₁) coupling with proton transport. Replacement of βSer174 by Phe in β-sheet4 of the β subunit (βS174F) caused slow γ subunit revolution of the F₁ sector, consistent with the decreased ATPase activity [M. Nakanishi-Matsui, S. Kashiwagi, T. Ubukata, A. Iwamoto-Kihara, Y. Wada, M. Futai, Rotational catalysis of Escherichia coli ATP synthase F1 sector. Stochastic fluctuation and a key domain of the β subunit, J. Biol. Chem. 282 (2007) 20698-20704]. Modeling of the domain including β-sheet4 and α-helixB predicted that the mutant βPhe174 residue undergoes strong and weak hydrophobic interactions with βIle163 and βIle166, respectively. Supporting this prediction, the replacement of βIle163 in α-helixB by Ala partially suppressed the βS174F mutation: in the double mutant, the revolution speed and ATPase activity recovered to about half of the levels in the wild-type. Replacement of βIle166 by Ala lowered the revolution speed and ATPase activity to the same levels as in βS174F. Consistent with the weak hydrophobic interaction, βIle166 to Ala mutation did not suppress βS174F. Importance of the hinge domain [phosphate-binding loop (P-loop)/α-helixB/loop/β-sheet4, βPhe148-βGly186] as to driving rotational catalysis is discussed.     

Met23Lys mutation in subunit gamma of FOF1-ATP synthase from Rhodobacter capsulatus impairs the activation of ATP hydrolysis by protonmotive force

H⁺-F_OF₁-ATP synthase couples proton flow through its membrane portion, F_O, to the synthesis of ATP in its headpiece, F₁. Upon reversal of the reaction the enzyme functions as a proton pumping ATPase. Even in the simplest bacterial enzyme the ATPase activity is regulated by several mechanisms, involving inhibition by MgADP, conformational transitions of the ε subunit, and activation by protonmotive force. Here we report that the Met23Lys mutation in the γ subunit of the Rhodobacter capsulatus ATP synthase significantly impaired the activation of ATP hydrolysis by protonmotive force. The impairment in the mutant was due to faster enzyme deactivation that was particularly evident at low ATP/ADP ratio. We suggest that the electrostatic interaction of the introduced γLys23 with the DELSEED region of subunit β stabilized the ADP-inhibited state of the enzyme by hindering the rotation of subunit γ rotation which is necessary for the activation.     

Defective assembly of a hybrid vacuolar H-ATPase containing the mouse testis-specific E1 isoform and yeast subunits

Mammalian vacuolar-type proton pumping ATPases (V-ATPases) are diverse multi-subunit proton pumps. They are formed from membrane V_o and catalytic V₁ sectors, whose subunits have cell-specific or ubiquitous isoforms. Biochemical study of a unique V-ATPase is difficult because ones with different isoforms are present in the same cell. However, the properties of mouse isoforms can be studied using hybrid V-ATPases formed from the isoforms and other yeast subunits. As shown previously, mouse subunit E isoform E1 (testis-specific) or E2 (ubiquitous) can form active V-ATPases with other subunits of yeast, but E1/yeast hybrid V-ATPase is defective in proton transport at 37 °C (Sun-Wada, G.-H., Imai-Senga, Y., Yamamoto, A., Murata, Y., Hirata, T., Wada, Y., and Futai, M., 2002, J. Biol. Chem. 277, 18098-18105). In this study, we have analyzed the properties of E1/yeast hybrid V-ATPase to understand the role of the E subunit. The proton transport by the defective hybrid ATPase was reversibly recovered when incubation temperature of vacuoles or cells was shifted to 30 °C. Corresponding to the reversible defect of the hybrid V-ATPase, the V_o subunit a epitope was exposed to the corresponding antibody at 37 °C, but became inaccessible at 30 °C. However, the V₁ sector was still associated with V_o at 37 °C, as shown immunochemically. The control yeast V-ATPase was active at 37 °C, and its epitope was not accessible to the antibody. Glucose depletion, known to dissociate V₁ from V_o in yeast, had only a slight effect on the hybrid at acidic pH. The domain between Lys26 and Val83 of E1, which contains eight residues not conserved between E1 and E2, was responsible for the unique properties of the hybrid. These results suggest that subunit E, especially its amino-terminal domain, plays a pertinent role in the assembly of V-ATPase subunits in vacuolar membranes.     

Identification of amino acid residues essential for reconstitutive activity of subunit IV of the cytochrome bc1 complex from Rhodobacter sphaeroides

A region of subunit IV comprising residues 77-85 is identified as essential for interaction with the core complex to restore the bc₁ activity (reconstitutive activity). Recombinant subunit IV mutants with single or multiple alanine substitution at this region were generated and characterized to identify the essential amino acid residues. Residues 81-84, with sequence of YRYR, are required for reconstitutive activity of subunit IV, because a mutant with these four residues substituted with alanine has little activity, while a mutant with alanine substitution at residues 77-80 and 85 have the same reconstitutive activity as that of the wild-type IV. The positively charged group at R-82 and R-84 and both the hydroxyl group and aromatic group at Y-81 and Y-83 are essential. The interactions between these four residues of subunit IV and residues of core subunits are also responsible for the stability of the complex. However, these interactions are not essential for the incorporation of subunit IV into the bc₁ complex.     

In silico and in vitro characterization of phospholipase A2 isoforms from soybean (Glycine max)

At the present, no secreted phospholipase A₂ (sPLA₂) from soybean (Glycine max) was investigated in detail. In this work we identified five sequences of putative secreted sPLA₂ from soybean after a BLAST search in G. max database. Sequence analysis showed a conserved PA2c domain bearing the Ca²⁺ binding loop and the active site motif. All the five mature proteins contain 12 cysteine residues, which are commonly conserved in plant sPLA₂s. We propose a phylogenetic tree based on sequence alignment of reported plant sPLA₂s including the novel enzymes from G. max. According to PLA₂ superfamily, two of G. max sPLA₂s are grouped as XIA and the rest of sequences as XIB, on the basis of differences found in their molecular weights and deviating sequences especially in the N- and C-terminal regions of the isoenzymes. Furthermore, we report the cloning, expression and purification of one of the putative isoenzyme denoted as GmsPLA₂-XIA-1. We demonstrate that this mature sPLA₂ of 114 residues had PLA₂ activity on Triton:phospholipid mixed micelles and determine the kinetic parameters for this system. We generate a model based on the known crystal structure of sPLA₂ from rice (isoform II), giving first insights into the three-dimensional structure of folded GmsPLA₂-XIA-1. Besides describing the spatial arrangement of highly conserved pair HIS-49/ASP-50 and the Ca⁺² loop domains, we propose the putative amino acids involved in the interfacial recognition surface. Additionally, molecular dynamics simulations indicate that calcium ion, besides its key function in the catalytic cycle, plays an important role in the overall stability of GmsPLA₂-XIA-1 structure.     

Structural model of the transmembrane Fo rotary sector of H-transporting ATP synthase derived by solution NMR and intersubunit cross-linking in situ

H⁺-transporting, F₁F_o-type ATP synthases utilize a transmembrane H⁺ potential to drive ATP formation by a rotary catalytic mechanism. ATP is formed in alternating β subunits of the extramembranous F₁ sector of the enzyme, synthesis being driven by rotation of the γ subunit in the center of the F₁ molecule between the alternating catalytic sites . The H⁺ electrochemical potential is thought to drive γ subunit rotation by first coupling H⁺ transport to rotation of an oligomeric rotor of c subunits within the transmembrane F_o sector. The γ subunit is forced to turn with the c-oligomeric rotor due to connections between subunit c and the γ and ε subunits of F₁. In this essay we will review recent studies on the Escherichia coli F_o sector. The monomeric structure of subunit c, determined by NMR, shows that subunit c folds in a helical hairpin with the proton carrying Asp⁶¹ centered in the second transmembrane helix (TMH). A model for the structural organization of the c₁₀ oligomer in F_o was deduced from extensive cross-linking studies and by molecular modeling. The model indicates that the H⁺-carrying carboxyl of subunit c is occluded between neighboring subunits of the c₁₀ oligomer and that two c subunits pack in a “front-to-back” manner to form the H⁺ (cation) binding site. In order for protons to gain access to Asp⁶¹ during the protonation/deprotonation cycle, we propose that the outer, Asp⁶¹-bearing TMH-2s of the c-ring and TMHs from subunits composing the inlet and outlet channels must turn relative to each other, and that the swiveling motion associated with Asp⁶¹ protonation/deprotonation drives the rotation of the c-ring. The NMR structures of wild-type subunit c differs according to the protonation state of Asp⁶¹. The idea that the conformational state of subunit c changes during the catalytic cycle is supported by the cross-linking evidence in situ, and two recent NMR structures of functional mutant proteins in which critical residues have been switched between TMH-1 and TMH-2. The structural information is considered in the context of the possible mechanism of rotary movement of the c₁₀ oligomer during coupled synthesis of ATP.     

H2O2 induces rapid biophysical and permeability changes in the plasma membrane of Saccharomyces cerevisiae

In Saccharomyces cerevisiae, the diffusion rate of hydrogen peroxide (H₂O₂) through the plasma membrane decreases during adaptation to H₂O₂ by means of a mechanism that is still unknown. Here, evidence is presented that during adaptation to H₂O₂ the anisotropy of the plasma membrane increases. Adaptation to H₂O₂ was studied at several times (15min up to 90min) by applying the steady-state H₂O₂ delivery model. For wild-type cells, the steady-state fluorescence anisotropy increased after 30min, or 60min, when using 2-(9-anthroyloxy) stearic acid (2-AS), or diphenylhexatriene (DPH) membrane probe, respectively. Moreover, a 40% decrease in plasma membrane permeability to H₂O₂ was observed at 15min with a concomitant two-fold increase in catalase activity. Disruption of the ergosterol pathway, by knocking out either ERG3 or ERG6, prevents the changes in anisotropy during H₂O₂ adaptation. H₂O₂ diffusion through the plasma membrane in S. cerevisiae cells is not mediated by aquaporins since the H₂O₂ permeability constant is not altered in the presence of the aquaporin inhibitor mercuric chloride. Altogether, these results indicate that the regulation of the plasma membrane permeability towards H₂O₂ is mediated by modulation of the biophysical properties of the plasma membrane.     

Crystallographic and enzymatic insights into the mechanisms of Mg-ADP inhibition in the A1 complex of the A1AO ATP synthase

F-ATP synthases are described to have mechanisms which regulate the unnecessary depletion of ATP pool during an energy limited state of the cell. Mg-ADP inhibition is one of the regulatory features where Mg-ADP gets entrapped in the catalytic site, preventing the binding of ATP and further inhibiting ATP hydrolysis. Knowledge about the existence and regulation of the related archaeal-type A₁A_O ATP synthases (A₃B₃CDE₂FG₂ac) is limited. We demonstrate MgADP inhibition of the enzymatically active A₃B₃D- and A₃B₃DF complexes of Methanosarcina mazei Gö1 A-ATP synthase and reveal the importance of the amino acids P235 and S238 inside the P-loop (GPFGSGKTV) of the catalytic A subunit. Substituting these two residues by the respective P-loop residues alanine and cysteine (GAFGCGKTV) of the related eukaryotic V-ATPase increases significantly the ATPase activity of the enzyme variant and abolishes MgADP inhibition. The atomic structure of the P235A, S238C double mutant of subunit A of the Pyrococcus horikoshii OT3 A-ATP synthase provides details of how these critical residues affect nucleotide-binding and ATP hydrolysis in this molecular engine. The qualitative data are confirmed by quantitative results derived from fluorescence correlation spectroscopy experiments.     

Disulfide linkage in the coiled-coil domain of subunit H of A1AO ATP synthase from Methanocaldococcus jannaschii and the NMR structure of the C-terminal segment H85-104

The C-terminal residues 98-104 are important for structure stability of subunit H of A₁A_O ATP synthases as well as its interaction with subunit A. Here we determined the structure of the segment H_85-104 of H from Methanocaldococcus jannaschii, showing a helix between residues Lys90 to Glu100 and flexible tails at both ends. The helix-helix arrangement in the C-terminus was investigated by exchange of hydrophobic residues to single cysteine in mutants of the entire subunit H (H_I93C, H_L96C and H_L98C). Together with the surface charge distribution of H_85-104, these results shine light into the A-H assembly of this enzyme.