Crystal structure of A3B3 complex of V-ATPase from Thermus thermophilus
2009; Springer Nature; Volume: 28; Issue: 23 Linguagem: Inglês
10.1038/emboj.2009.310
ISSN1460-2075
AutoresMegan J. Maher, Satoru Akimoto, Momi Iwata, Koji Nagata, Y. Hori, Masasuke Yoshida, Shigeyuki Yokoyama, So Iwata, Ken Yokoyama,
Tópico(s)Photosynthetic Processes and Mechanisms
ResumoArticle5 November 2009Open Access Crystal structure of A3B3 complex of V-ATPase from Thermus thermophilus Megan J Maher Megan J Maher Division of Molecular Biosciences, Imperial College London, South Kensington Campus, London, UKPresent address: Centenary Institute, Locked Bag No. 6, Newtown, New South Wales 2042, Australia Search for more papers by this author Satoru Akimoto Satoru Akimoto Protein Research Group, Genomic Sciences Center, Yokohama Institute, RIKEN, 1-7-22 Suehiro-cho, Tsurumi-ku, Yokohama, Japan Search for more papers by this author Momi Iwata Momi Iwata Division of Molecular Biosciences, Imperial College London, South Kensington Campus, London, UK Membrane Protein Laboratory, Diamond Light Source Limited, Harwell Science and Innovation Campus, Chilton, Didcot, Oxfordshire, UK Search for more papers by this author Koji Nagata Koji Nagata Division of Molecular Biosciences, Imperial College London, South Kensington Campus, London, UK Search for more papers by this author Yoshiko Hori Yoshiko Hori Protein Research Group, Genomic Sciences Center, Yokohama Institute, RIKEN, 1-7-22 Suehiro-cho, Tsurumi-ku, Yokohama, Japan Search for more papers by this author Masasuke Yoshida Masasuke Yoshida Chemical Resources Laboratory, Tokyo Institute of Technology, Midori-ku, Yokohama, Japan ICORP, ATP Synthesis Regulation Project, Japan Science and Technology Agency, National Museum of Emerging Science and Innovation, Koto-ku, Tokyo, Japan Search for more papers by this author Shigeyuki Yokoyama Shigeyuki Yokoyama Protein Research Group, Genomic Sciences Center, Yokohama Institute, RIKEN, 1-7-22 Suehiro-cho, Tsurumi-ku, Yokohama, Japan Search for more papers by this author So Iwata Corresponding Author So Iwata Division of Molecular Biosciences, Imperial College London, South Kensington Campus, London, UK Membrane Protein Laboratory, Diamond Light Source Limited, Harwell Science and Innovation Campus, Chilton, Didcot, Oxfordshire, UK Department of Cell Biology, Faculty of Medicine, Kyoto University, Yoshidakonoe-cho, Sakyo-ku, Kyoto, Japan Human Receptor Crystallography Project, Exploratory Research for Advanced Technology (ERATO), Japan Science and Technology Agency, Yoshidakonoe-cho, Sakyo-ku, Kyoto, Japan Search for more papers by this author Ken Yokoyama Corresponding Author Ken Yokoyama Protein Research Group, Genomic Sciences Center, Yokohama Institute, RIKEN, 1-7-22 Suehiro-cho, Tsurumi-ku, Yokohama, Japan Chemical Resources Laboratory, Tokyo Institute of Technology, Midori-ku, Yokohama, Japan ICORP, ATP Synthesis Regulation Project, Japan Science and Technology Agency, National Museum of Emerging Science and Innovation, Koto-ku, Tokyo, Japan Search for more papers by this author Megan J Maher Megan J Maher Division of Molecular Biosciences, Imperial College London, South Kensington Campus, London, UKPresent address: Centenary Institute, Locked Bag No. 6, Newtown, New South Wales 2042, Australia Search for more papers by this author Satoru Akimoto Satoru Akimoto Protein Research Group, Genomic Sciences Center, Yokohama Institute, RIKEN, 1-7-22 Suehiro-cho, Tsurumi-ku, Yokohama, Japan Search for more papers by this author Momi Iwata Momi Iwata Division of Molecular Biosciences, Imperial College London, South Kensington Campus, London, UK Membrane Protein Laboratory, Diamond Light Source Limited, Harwell Science and Innovation Campus, Chilton, Didcot, Oxfordshire, UK Search for more papers by this author Koji Nagata Koji Nagata Division of Molecular Biosciences, Imperial College London, South Kensington Campus, London, UK Search for more papers by this author Yoshiko Hori Yoshiko Hori Protein Research Group, Genomic Sciences Center, Yokohama Institute, RIKEN, 1-7-22 Suehiro-cho, Tsurumi-ku, Yokohama, Japan Search for more papers by this author Masasuke Yoshida Masasuke Yoshida Chemical Resources Laboratory, Tokyo Institute of Technology, Midori-ku, Yokohama, Japan ICORP, ATP Synthesis Regulation Project, Japan Science and Technology Agency, National Museum of Emerging Science and Innovation, Koto-ku, Tokyo, Japan Search for more papers by this author Shigeyuki Yokoyama Shigeyuki Yokoyama Protein Research Group, Genomic Sciences Center, Yokohama Institute, RIKEN, 1-7-22 Suehiro-cho, Tsurumi-ku, Yokohama, Japan Search for more papers by this author So Iwata Corresponding Author So Iwata Division of Molecular Biosciences, Imperial College London, South Kensington Campus, London, UK Membrane Protein Laboratory, Diamond Light Source Limited, Harwell Science and Innovation Campus, Chilton, Didcot, Oxfordshire, UK Department of Cell Biology, Faculty of Medicine, Kyoto University, Yoshidakonoe-cho, Sakyo-ku, Kyoto, Japan Human Receptor Crystallography Project, Exploratory Research for Advanced Technology (ERATO), Japan Science and Technology Agency, Yoshidakonoe-cho, Sakyo-ku, Kyoto, Japan Search for more papers by this author Ken Yokoyama Corresponding Author Ken Yokoyama Protein Research Group, Genomic Sciences Center, Yokohama Institute, RIKEN, 1-7-22 Suehiro-cho, Tsurumi-ku, Yokohama, Japan Chemical Resources Laboratory, Tokyo Institute of Technology, Midori-ku, Yokohama, Japan ICORP, ATP Synthesis Regulation Project, Japan Science and Technology Agency, National Museum of Emerging Science and Innovation, Koto-ku, Tokyo, Japan Search for more papers by this author Author Information Megan J Maher1,‡, Satoru Akimoto2,‡, Momi Iwata1,3,‡, Koji Nagata1, Yoshiko Hori2, Masasuke Yoshida4,5, Shigeyuki Yokoyama2, So Iwata 1,3,6,7 and Ken Yokoyama 2,4,5 1Division of Molecular Biosciences, Imperial College London, South Kensington Campus, London, UK 2Protein Research Group, Genomic Sciences Center, Yokohama Institute, RIKEN, 1-7-22 Suehiro-cho, Tsurumi-ku, Yokohama, Japan 3Membrane Protein Laboratory, Diamond Light Source Limited, Harwell Science and Innovation Campus, Chilton, Didcot, Oxfordshire, UK 4Chemical Resources Laboratory, Tokyo Institute of Technology, Midori-ku, Yokohama, Japan 5ICORP, ATP Synthesis Regulation Project, Japan Science and Technology Agency, National Museum of Emerging Science and Innovation, Koto-ku, Tokyo, Japan 6Department of Cell Biology, Faculty of Medicine, Kyoto University, Yoshidakonoe-cho, Sakyo-ku, Kyoto, Japan 7Human Receptor Crystallography Project, Exploratory Research for Advanced Technology (ERATO), Japan Science and Technology Agency, Yoshidakonoe-cho, Sakyo-ku, Kyoto, Japan ‡These authors contributed equally to this work *Corresponding authors: Division of Molecular Biosciences, Imperial College London, South Kensington Campus, London SW7 2AZ, UK. Tel.: +44 (0)20 7594 3064; Fax: +44 (0)20 7594 3065; E-mail: [email protected] Resource Laboratory, Tokyo Institute of Technology, Nagatsuda 4259, Midori-ku, Yokohama 226-8503, Japan. Tel.: +81 45 924 5267; Fax: +81 45 924 5277; E-mail: [email protected] The EMBO Journal (2009)28:3771-3779https://doi.org/10.1038/emboj.2009.310 Present address: Centenary Institute, Locked Bag No. 6, Newtown, New South Wales 2042, Australia PDFDownload PDF of article text and main figures. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Vacuolar-type ATPases (V-ATPases) exist in various cellular membranes of many organisms to regulate physiological processes by controlling the acidic environment. Here, we have determined the crystal structure of the A3B3 subcomplex of V-ATPase at 2.8 Å resolution. The overall construction of the A3B3 subcomplex is significantly different from that of the α3β3 sub-domain in FoF1-ATP synthase, because of the presence of a protruding ‘bulge’ domain feature in the catalytic A subunits. The A3B3 subcomplex structure provides the first molecular insight at the catalytic and non-catalytic interfaces, which was not possible in the structures of the separate subunits alone. Specifically, in the non-catalytic interface, the B subunit seems to be incapable of binding ATP, which is a marked difference from the situation indicated by the structure of the FoF1-ATP synthase. In the catalytic interface, our mutational analysis, on the basis of the A3B3 structure, has highlighted the presence of a cluster composed of key hydrophobic residues, which are essential for ATP hydrolysis by V-ATPases. Introduction The vacuolar-type ATPases/synthases (V-ATPases) are widely distributed in many organisms and are involved in a variety of physiological processes (Forgac, 2007). The eukaryotic V-ATPases function as proton pumps that are involved in the acidification of cellular compartments such as the Golgi apparatus or lysosomes. In addition, some prokaryotes, such as the thermophilic eubacterium, Thermus thermophilus, contain a member of the family (or homologue) of the V-ATPases in their membranes (Yokoyama et al, 1990; Yokoyama and Imamura, 2005). The prokaryotic V-ATPase is made up of a simpler subunit composition than the eukaryotic enzymes, but each subunit shows significant sequence similarity to its eukaryotic counterpart (Tsutsumi et al, 1991; Yokoyama et al, 2003). For instance, the A subunit of T. thermophilus shows 49% sequence identity and 67% similarity to its human counterpart (Supplementary Figure S1A). The V-ATPases and FoF1-ATPases (FoF1), which function as ATP synthases in mitochondoria, chloroplast and most bacteria, are evolutionarily related and use a similar rotary catalytic mechanism that couples ATP hydrolysis/synthesis in the water soluble part of F1 or V1 with proton translocation across the membranes in the hydrophobic domain of Fo or Vo (Yoshida et al, 2001; Forgac, 2007). The isolated Escherichia coli F1, which has the subunit stoichiometry α3β3γ1δ1ε1, hydrolyses ATP with high cooperativity between the three catalytic sites contained in the β subunits at the α/β interfaces. The crystal structure of bovine F1 revealed remarkable asymmetry in the conformation and nucleotide occupancy at the α–β interfaces, consistent with the binding change model for rotary catalysis (Boyer, 1993; Abrahams et al, 1994). In the binding change model, a central γ subunit is surrounded by an α3β3 cylinder, with the β subunits at three catalytic interfaces bearing an ATP or ADP analogue. In this structure, two of the β subunits (βD, βT) at catalytic interfaces bearing nucleotides are in closed conformations, and the third β subunit, which is free of bound nucleotides (βE), has an open conformation. Sequential conformation change of each β subunit (closed to open) during turnover is coupled to rotation of the γ subunit. In reverse, rotation of the γ subunit, which is driven by the proton motive force, drives sequential conformational changes in the β subunits, resulting in ADP phosphorylation and release of product ATP from the enzyme. Analysis of single particles by electron microscopy has indicated that the overall features of V1 are nearly identical to those of F1. The A and B subunits also share ∼25% amino-acid sequence identity with the β and α subunits of F1, respectively. In particular, the ATP-binding regions of the catalytic A subunits in V1 and the β subunits in F1 are highly conserved. However, sequence alignment of the A subunit with the β subunit revealed that an additional non-homologus region is present in the A subunit of V1 (Forgac, 2007). Earlier mutagenesis studies have suggested that the non-homologous region likely has an important function in energy coupling between the V1 and Vo moieties (Shao et al, 2003). The V1 of T. thermophilus is composed of an A3B3 cylinder and the D and F subunits (Yokoyama and Imamura, 2005). Experimental evidence for rotation in V1 has been obtained by direct visualization of polystyrene beads attached to the D or F subunit, indicating that V1 catalyses ATP hydrolysis using a binding change mechanism similar to that of F1 (Imamura et al, 2003, 2005). On the basis of analogy with F1 and several mutagenesis studies on V-ATPases, it has been proposed that ATP hydrolysis occurs at three catalytic sites sequentially, each located at the ‘B–A interface’, one of two interfaces between the A and B subunits (Yokoyama and Imamura, 2005; Forgac, 2007). Most residues in this site, including a P-loop motif responsible for nucleotide binding, are from the A subunit. The other interface, between the A and B subunits, is called the ‘A–B interface’. The P-loop motif is not conserved in the B subunit and the isolated B subunit of T. thermophilus V-ATPase did not bind nucleotides, unlike the isolated A subunit (Yokoyama et al, 1998; Imamura et al, 2006). A reactive ATP analogue was, however, shown to modify the B subunit, which has been interpreted as possible nucleotide binding to the A–B interface (Manolson et al, 1985; Vasilyeva and Forgac, 1996). Here, we present the X-ray structure of the V-ATPase A3B3 complex from T. thermophilus. The overall structure shows clear differences from that of the α3β3 sub-domain in F1 because of the presence of a protruding ‘bulge’ domain, belonging to the A subunit. Comparisons with the F1 structure, in addition to mutagenesis studies at the B–A and A–B interfaces, have highlighted the importance of some hydrophobic residues in catalysis. We have also re-examined the reported nucleotide binding to the B subunits (supported by chemical modification), using a reactive ATP analogue. Our X-ray structure also reveals many similarities and differences between the F1 and V1 at the catalytic interface, which contribute to a further understanding of the rotary catalytic mechanism of theses ATPases. Results and discussion Structure determination of the A3B3 subcomplex The A3B3 complex, which does not contain nucleotides, was prepared as described earlier. The mutant A3B3 complex, in which all cysteine residues (A/C28, C255, C508, B/C265) were substituted with serine residues, was used for crystallization. The structure of A3B3 was solved by molecular replacement, using partial structures of the A and B subunits from archea (PDB codes 1VDZ and 2C61, respectively) as search models. The crystallographic asymmetric unit contains two AB dimers, which are virtually structurally identical. Extensive remodelling was required for both the A and B subunit structures, because the isolated subunit structures were considerably different from those in the complex, particularly in the region of the subunit interfaces (Supplementary Figure S2). Accordingly, the A3B3 complex structure provides much new information about the structures of the subunit interfaces, which are described below. The structure was refined using the data up to 2.8 Å resolution. The data collection and refinement statistics are summarized in Table I. A small fraction of the crystal (15%) is twinned with a twinning operator of (−h, −k, l) and this contribution was included during the final refinement of the model (Supplementary Figure S3). The average B-factors for the N-terminal, the bulge, the central α/β and the C-terminal domains of the A subunit are 18.0/18.0, 22.5/22.4, 29.6/29.6 and 46.3/46.3 Å2, respectively (two numbers represent the values for two different A subunits in the asymmetric unit). The average B-factors for the N-terminal, the central α/β and the C-terminal domains of the B subunit are 20.2/20.2, 29.1/29.1 and 46.5/46.5 Å2, respectively. As indicated by the average B-factors, the C-terminal domains of the both A and B subunits are rather disordered and it was not possible to model side chains accurately. Other details are described in Materials and methods. Table 1. Summary of data collection and refinement Native Data collection statistics Resolution (Å) 20–2.8 (2.9–2.8)a Wavelength (Å) 0.933 Number of reflections Overall 251 463 Unique 98 563 (9568)a Completeness (%) 97.7 (96.1)a I/σ 11.6 (2.02)a Rmerge (%)b 8.7 (43)a Refinement statistics Residue range A1–A578, B5–B464, C1–C578, D5–D464 Number of non-hydrogen atoms 16 863 Number of water molecules 707 Average temperature factor (Å2) 30.85 Resolution range (Å) 20–2.8 (2.87–2.8)a Twin fractions (h, k, l) 0.85/(−h, −k, l) 0.15 Rcryst (%)c 25.0 (28.3)a Without de-twinning 27.2 (34.1)a Number of reflections 93 409 (4990)a Rfree (%)d 28.1 (33.5)a Without de-twinning 32.5 (39.1)a Number of reflections 6485 (312)a Estimated overall coordinate error (Å) 0.122 RMS deviations from ideal values Bond length (Å) 0.011 Bond angles (deg) 1.407 Torsion angles (deg) 6.478 Chiral centre restraints (Å3) 0.092 General planes (Å) 0.006 Ramachandran plot (non-Gly, non-Pro residues) Residues in favoured and allowed regions (%) 99.8 Residues in disallowed regions (%) 0.1 a Values for the highest resolution shell are given in parentheses. b Rmerge=∑h∑i∣Ii(h)−〈I(h)〉∣/∑h∑i(h), where Ii(h) is the ith measurement. c Rcryst=∑∥Fo∣−∣Fc∥/∑∣Fo∣, where Fo and Fc are observed and calculated structure factors, respectively. d Rfree: The R-factor as defined above, but calculated using a subset (5%) of reflections that are not used in the refinement. Overall structure Figure 1 shows the top (A) and side (B) views of the A3B3 complex crystal structure. The three catalytic A subunits and three catalytic B subunits are arranged alternately as in F1. Both A and B subunits are nucleotide free in the crystal structure. The six A and B subunits in the complex are related by an approximate six-fold symmetry, which is highlighted by the N-terminal β-barrel domains (shown as blue ribbons). This six-fold symmetry, however, breaks down because of protruding structures from the A subunits (shown as red ribbons), which makes the A3B3 complex a triangular shape in the top view. This protruding structure, referred to as the bulge hereafter, is encoded by the ‘non-homologus region’ of the A subunit that is not conserved in F1. In the side view, perpendicular to the molecular three-fold axis, the A3B3 complex structure does not have the orange-like spherical shape of the F1 (Abrahams et al, 1994). The main structural differences between the V-ATPase A3B3 complex and the α3β3 sub-domain in F1 are observed in two regions in the A subunit. One is the bulge mentioned above and the other is the presence of three longer helices in the C-terminal domain facing the Vo domain in the membrane. The A3B3 complex maintains a large internal cavity, which should accommodate a part of the DF central shaft in the full complex. The diameter of the cavity is larger than those observed for the α3β3 sub-domain in F1 because all A subunits in the complex are present in an ‘open’ conformation as described below. Figure 1.Crystal structure of the A3B3 complex. (A) Top view of the complex viewed towards the membrane. The A and B subunits are circled by magenta and green lines, respectively. (B) Side view of the A3B3 complex orthogonal to the three-fold axis. The N-terminal β-barrel domains, the α/β domains and the C-terminal domains from the A and B subunits are shown in blue, light grey and green, respectively. The bulge domain and the extended linker from the A subunits are shown in red and purple, respectively. The catalytic and non-catalytic interfaces, the P-loop, the hydrophobic loop and two important amino-acid residues in the A3B3 complex are schematically represented in the lower panels of the figure. Download figure Download PowerPoint B subunit structure and the non-catalytic A–B interface The B subunit in the A3B3 complex consists of the N-terminal β-barrel domain (5–79), the central α/β domain (80–373) and the C-terminal domain (374–464), as shown in Figure 2. Secondary structure elements of A and B subunits are summarized in Supplementary Figure S4. The B subunit has been suggested to be the non-catalytic subunit, equivalent to the F1-α subunit (Manolson et al, 1985; Vasilyeva and Forgac, 1996; Forgac, 2007). This was examined by comparing the B subunit structure with α and β subunit structures of yeast F1, respectively (Kabaleeswaran et al, 2006; PDB ID: 2HLD). In the F1 structure, all three α subunits adopt similar conformations, whereas the β subunits are in three distinct conformations, namely βD, βT and βE (Abrahams et al, 1994). Using the Cα atom positions, the whole B subunit can be superimposed onto the best fit α and β (βE) subunits with r.m.s.d. values of 1.7 and 1.9 Å, respectively, when the molecule 2 of 2HLD is used. This means that the nucleotide-free B subunit structure is more similar to the α subunit structure with bound AMPPMP than to the nucleotide-free β subunit (βE) structure. The similarity of these two subunits is highlighted when their domains are compared. The N-terminal β-barrel, central α/β and C-terminal domains of the B subunit can be superimposed onto the respective domains of the α subunit, with r.m.s.ds of 1.7, 1.3 and 1.6 Å, respectively. Figure 2.Structures of the A and B subunits in the complex. (A) A subunit structure. The β-barrel (A1–72) and the C-terminal (A442–565) domains are shown in blue and green, respectively. The bulge domain (A113–184), the extended linker (A185–204), the P-loop (A222–250) and the hydrophobic loop (A374–407) are highlighted in red, purple, magenta and cyan, respectively. The conserved proline residues in the extended linker are shown in purple and labelled. These proline residues have been reported to be important for the coupling efficiency of yeast V-ATPase (Shao et al, 2003). (B) B subunit structure. The β-barrel (B5–79) and the C-terminal (B374–464) domains are shown in blue and green, respectively. Tyrosine 331 in the B subunit is shown in sky blue. (C) A subunit (red) superimposed on the βE subunit of the yeast F1. (D) A subunit (red) superimposed on the βD (blue) subunit of the yeast F1. Download figure Download PowerPoint It has been thought that the B subunit could contain a nucleotide-binding site because it is modified by the ATP analogue of 3-O-(4-benzoyl) benzoyladenosine 5′-triphosphate (Bz-ATPase) or 2-azido-ATP (Manolson et al, 1985). On the other hand, the structure of the potential nucleotide-binding site of the B subunit in the A–B interface is very different from that observed in the β–α interface of F1, which binds a non-catalytic nucleotide. Unlike the non-catalytic α subunit, the P-loop sequence (GXXXXGKT/S, also known as ‘Walker motif A’) is not conserved in the B subunit. Figure 3 compares the structures of the P-loop equivalent region in the B subunit and of the nucleotide-bound P-loop in the yeast F1 αE subunit. The P-loop has a unique main-chain structure, which allows five amide protons within the loop to form hydrogen-bonding interactions (shown in blue in Figure 3B) with the triphosphate group of bound ATP. The equivalent loop in the B subunit shows a totally different main-chain conformation, which by analogy does not allow the formation of these hydrogen bonds (Figure 3A and C). This is presumably because the P-loop equivalent region in the B subunit lacks flexible glycine residues and includes an insertion of a proline residue, which does not have an amide group. In addition, several residues known to be involved in nucleotide binding at the β–α interface (β/Y368, β/R372, α/Q172, α/Q432, α/P367, the bovine F1 residue numbering is used hereafter) are missing in the A3B3 complex. In the F1 structure, the side chains of α/Q172, α/K175, α/T176 in the P-loop region interact with the diphosphate group of ATP-Mg at the βT–αE interface. Substitution of these residues with alanine causes the complete loss of ATP binding to the α subunit, resulting in a dramatic loss of steady-state ATP hydrolytic activity (Matsui et al, 1997). In contrast, a double mutant of V1 (B/N161/A (equivalent to α/T176) and B/E162A (equivalent to α/A177)), which eliminates hydrophilic residues on the loop region, shows a comparable ATP-hydrolysis rate to that of the wild-type V1 (Table II). These mutations did not affect the profile of ATP hydrolysis of V1 either (Supplementary Figure S6). In conclusion, it is unlikely that the B subunit binds a nucleotide at the A–B interface in a similar manner to that of the α subunit. The chemical modification of the B subunit by nucleotide analogues can be explained as a modification of the B subunit residues in the catalytic B–A interface, but not in the A–B interface. As discussed below, we found a conserved B/Y331 at the nucleotide-binding site in the B–A interface, which is the likely target of this modification. Figure 3.Structures of the P-loop equivalent region in the B subunit and of the nucleotide-bound P-loop in the yeast F1 αE subunit. (A) Structure of the P-loop equivalent region in the B subunit. (B) Structure of the P-loop of the yeast F1α subunit. The bound AMP-PNP molecule and the magnesium ion are shown. Hydrogen bonds are shown in light blue, whereas coordination links to the magnesium ion are shown in yellow. (C) Superposition of (A) and (B). Only protein atoms are shown. Download figure Download PowerPoint Table 2. Effect of mutagenesis on the ATP hydrolysis activity of V1/F1 V1 F1 Mutation Turnover rate (s−1) Relative activity (%) Mutation Turnover rate (s−1) Relative activity (%) Wt 108±13 100 Wt 321±13 100 A/E257D 4.26±0.29 3.9 β/Y311F 170±3.6 53.0 A/E257Q 0.19±0.04 <1.0 β/Y311S 1.2±0.5 <1.0 A/Y419Q 0.37±0.03 <1.0 β/Y311A 2.2±0.4 <1.0 B/R360K 0.14±0.02 <1.0 B/R360L 0.13±0.02 <1.0 B/Y331S 0.09±0.02 <1.0 B/Y331F 48.1±10.0 44.3 B/Y331L 0.49±0.08 <1.0 B/Y331A 0.03±0.01 <1.0 A/F230A 6.00±3.22 5.5 A/S385A 1.24±0.19 1.1 B/N161A-E162A 116.6±1.4 107 The ATP hydrolysis activities of each mutant enzyme were measured by the enzyme-coupling assay. Each value represents the average of 3–5 replicates. The V1 contains the TSSA mutation as described in Materials and methods. Note that the residue numbers of bacillus PS3 F1 are on the basis of the bovine F1 system. A subunit structure The structure of the A subunit is shown in Figure 2A. The A subunit consists of four domains: the N-terminal β-barrel domain (1–72), the non-homologus domain including the bulge (113–184), the central α/β domain (205–441) and the C-terminal domain (442–565). The A subunit is the catalytic subunit of the V-ATPases and is equivalent to the F1-β subunit. As shown in Figure 2C and Table II, structures of the A and the β subunits are similar except for the presence of an additional bulge of eight β-strands and a larger C-terminal α-helical bundle in the A subunit. The bulge is connected to the nucleotide-binding domain through an extended linker (A185–204), which starts in the space between the N-terminal and the bulge domains and runs down the surface of the A3B3 complex (Figure 2A; Supplementary Figure S4A). Both the bulge and this extended linker are encoded by the so-called ‘non-homologus’ region of sequence (Shao et al, 2003). Interestingly, the residues within this linker (A/P188, A/P194 and A/P204) are conserved among V-ATPases (Supplementary Figure S1A). Substitution of the equivalent residues in yeast V-ATPase results in a change in coupling efficiency (Shao et al, 2003). These results suggest that the linker region is involved in the interaction between the peripheral stalk (composed of the E and G subunits) and the A3B3 complex. The presence of longer helices, including α13–α16 in the C-terminal domain, distinguishes the domain structure from that of the equivalent F1-β subunit (Figure 2D; Supplementary Figure S3A). This structure is also conserved in the isolated A subunit of Pyrococcus horikoshii (Maegawa et al, 2006), indicating that this is a general feature observed among V-ATPase structures. The A subunit superimposes well onto the structure of the F1-βE subunit, but not onto those of the βD and βT subunits (Figure 2C and D). This is because of conformational differences around the nucleotide-binding site of nucleotide-free (βE) and nucleotide-bound β subunits (Menz et al, 2001). A similar conformational change should be induced in the A subunit by nucleotide binding and/or interaction with the central rotor. That is, we predict that the lower part of the nucleotide-binding domain (205–245, 405–441) will rotate upwards, together with the C-terminal domain (442–565), as one group. This conformational change should act to place side chains critical for catalysis into the correct positions and conformations for nucleotide binding, as we discuss in the following section. Regulatory disulphide bond formation between conserved cysteine residues in the A subunit (A/C261 and C539 in yeast V-ATPase) has been proposed (Feng and Forgac, 1994). The equivalent cysteine residues in the T. thermophilus A subunit are A/S232 and A/S539 (mutated from A/C539), respectively. In our open structure of A subunit, the distance between the hydroxyl oxygen atom of A/S232 and the sulfur atom of A/C507 is 26 Å apart. It is, therefore, unlikely that a disulphide bridge exists between them. However, it is possible that side chain of A/S232 might be proximate to that of A/C507 when the A subunit adopts the closed conformation. Disulphide bond formation between two different subunits is unlikely because the relevant residues are more than 60 Å apart. The catalytic B–A interface The nucleotide-binding sites are located in the interface between the B and A subunits. The site is mainly composed of the residues from the A subunit, but some from the B subunit are also essential. The nucleotide-binding domain of the A subunit shows high sequence similarity to that of F1, including the P-loop, which is responsible for coordination of phosphate moieties of ATP (Supplementary Figure S1A). Fig
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