FRINK Linked Data Fragments server

Matches in SemOpenAlex for { <https://semopenalex.org/work/W2433610117> ?p ?o ?g. }

• W2433610117 endingPage "1706" @default.
• W2433610117 startingPage "1694" @default.
• W2433610117 abstract "Article13 June 2016free access Transparent process Crystal structure of yeast V1-ATPase in the autoinhibited state Rebecca A Oot Rebecca A Oot Department of Biochemistry and Molecular Biology, SUNY Upstate Medical University, Syracuse, NY, USA Search for more papers by this author Patricia M Kane Patricia M Kane Department of Biochemistry and Molecular Biology, SUNY Upstate Medical University, Syracuse, NY, USA Search for more papers by this author Edward A Berry Edward A Berry Department of Biochemistry and Molecular Biology, SUNY Upstate Medical University, Syracuse, NY, USA Search for more papers by this author Stephan Wilkens Corresponding Author Stephan Wilkens orcid.org/0000-0002-2721-2789 Department of Biochemistry and Molecular Biology, SUNY Upstate Medical University, Syracuse, NY, USA Search for more papers by this author Rebecca A Oot Rebecca A Oot Department of Biochemistry and Molecular Biology, SUNY Upstate Medical University, Syracuse, NY, USA Search for more papers by this author Patricia M Kane Patricia M Kane Department of Biochemistry and Molecular Biology, SUNY Upstate Medical University, Syracuse, NY, USA Search for more papers by this author Edward A Berry Edward A Berry Department of Biochemistry and Molecular Biology, SUNY Upstate Medical University, Syracuse, NY, USA Search for more papers by this author Stephan Wilkens Corresponding Author Stephan Wilkens orcid.org/0000-0002-2721-2789 Department of Biochemistry and Molecular Biology, SUNY Upstate Medical University, Syracuse, NY, USA Search for more papers by this author Author Information Rebecca A Oot1, Patricia M Kane1, Edward A Berry1 and Stephan Wilkens 1 1Department of Biochemistry and Molecular Biology, SUNY Upstate Medical University, Syracuse, NY, USA *Corresponding author. Tel: +1 315 464 8703; E-mail: [email protected] The EMBO Journal (2016)35:1694-1706https://doi.org/10.15252/embj.201593447 AM PDF Peer ReviewDownload a summary of the editorial decision process including editorial decision letters, reviewer comments and author responses to feedback. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Abstract Vacuolar ATPases (V-ATPases) are essential proton pumps that acidify the lumen of subcellular organelles in all eukaryotic cells and the extracellular space in some tissues. V-ATPase activity is regulated by a unique mechanism referred to as reversible disassembly, wherein the soluble catalytic sector, V1, is released from the membrane and its MgATPase activity silenced. The crystal structure of yeast V1 presented here shows that activity silencing involves a large conformational change of subunit H, with its C-terminal domain rotating ~150° from a position near the membrane in holo V-ATPase to a position at the bottom of V1 near an open catalytic site. Together with biochemical data, the structure supports a mechanistic model wherein subunit H inhibits ATPase activity by stabilizing an open catalytic site that results in tight binding of inhibitory ADP at another site. Synopsis The proton-pumping vacuolar ATPase is regulated by reversible disassembly of subcomplexes V1 and V0. The crystal structure of V1 reveals the basis for autoinhibition and provides a mechanism for V-ATPase regulation. The crystal structure of autoinhibited yeast V1-ATPase was solved at 6.2–6.5 Å resolution. Autoinhibition of V1-ATPase activity involves a large-scale domain rotation (150°) of the H subunit. The conformational change in the H subunit appears to stabilize inhibitory ADP in one of the three catalytic sites. The structural changes observed in V1 upon enzyme disassembly reveal the mechanism of V-ATPase regulation. Introduction The vacuolar H+-ATPase (V-ATPase, V1Vo-ATPase) is a large multisubunit enzyme complex found in the endomembrane system of all eukaryotic cells where it acidifies the lumen of subcellular organelles including lysosomes, endosomes, the Golgi apparatus, and clathrin-coated vesicles (Forgac, 2007). V-ATPase function is essential for pH and ion homeostasis, protein trafficking, endocytosis, MTOR, and NOTCH signaling, as well as hormone secretion and neurotransmitter release. V-ATPase can also be found in the plasma membrane of polarized animal cells where it pumps protons out of the cell, a process required for bone remodeling, urine acidification, and sperm maturation. While complete loss of V-ATPase function in animals is embryonic lethal (Inoue et al, 1999), partial loss or hyperactivity of the enzyme has been associated with a wide spectrum of human diseases including osteoporosis (Thudium et al, 2012), deafness (Karet et al, 1999), renal tubular acidosis (Smith et al, 2000), diabetes (Sun-Wada et al, 2006), infertility (Brown et al, 1997), neurodegeneration (Williamson & Hiesinger, 2010), and cancer (Sennoune et al, 2004), making V-ATPase a valuable drug target (Bowman & Bowman, 2005; Fais et al, 2007; Kartner & Manolson, 2014). The V-ATPase couples ATP hydrolysis with the transport of protons across membranes using a rotary mechanism much like the related F- and A/V-type ATPases (Futai et al, 2012) (Fig 1A). In the V-ATPase from S. cerevisiae, a well-characterized model system for the enzyme from higher organisms, energy coupling requires the concerted action of fourteen different polypeptides that are organized into the ~640-kDa membrane extrinsic V1-ATPase (A3B3CDE3FG3H) (Kitagawa et al, 2008) and the ~330-kDa membrane integral Vo proton channel (ac8c′c″de) (Powell et al, 2000; Zhao et al, 2015). ATP hydrolysis on the catalytic V1 is coupled with proton pumping across the Vo via a central rotor made of V1 and Vo subunits DFc8c′c″d. A peripheral stator complex composed of V1 subunits E,G,H,C serves to stabilize the motor by binding to the N-terminal cytosolic domain (aNT) of the membrane-anchored a subunit of the Vo (Fig 1B). Figure 1. Schematic of the bacterial and eukaryotic V-ATPases Bacterial sodium pumping A/V-type ATPase from E. hirae (subunit nomenclature of the eukaryotic V-ATPase). Note that the bacterial A/V-type enzyme has only two peripheral stalks (EG1 and EG2) and no equivalents for the C and H subunits found exclusively in eukaryotic V-ATPase (see next panel). Eukaryotic proton pumping V-ATPase. The eukaryotic V-ATPase is a dedicated proton pump composed of a soluble catalytic sector (V1) and a membrane integral proton translocating sector (Vo). The two functional sectors are linked via a peripheral stator that is formed from the N- terminal extension of the 100-kDa membrane integral subunit (aNT), three peripheral stalks (EG1-3; shown in blue/orange), and subunits C (red) and H (yellow) that are unique to the eukaryotic enzyme. Reversible disassembly of eukaryotic V-ATPase. Cellular signals, such as starvation, lead to a dramatic structural rearrangement wherein the V1 sector dissociates from Vo and subunit C is released from the enzyme. While dissociated, both the V1-ATPase and Vo proton translocation domain are functionally silenced. This process is fully reversible and upon reassembly, enzymatic activity is restored. Download figure Download PowerPoint As a major consumer of cellular energy, V-ATPase function must be tightly controlled. Regulation of enzyme activity is accomplished by a unique mechanism referred to as reversible dissociation, a condition under which the complex disassembles into cytoplasmic V1 and membrane-bound Vo (Kane, 1995; Sumner et al, 1995). As part of the process, the single-copy subunit C is released from the enzyme and re-incorporated during enzyme reassembly (Kane, 1995). Upon enzyme dissociation, V1 loses the ability to hydrolyze MgATP (Graf et al, 1996; Parra et al, 2000) and the Vo no longer conducts protons (Zhang et al, 1994), a phenomenon referred to as “activity silencing” (Fig 1C). Reversible dissociation of V-ATPase is well characterized in S. cerevisiae, but more recent data suggest that the mammalian enzyme is regulated by a similar process in some cell types. While yeast V-ATPase assembly is governed by environmental conditions such as nutrient availability (Parra & Kane, 1998), salinity, or pH (Diakov & Kane, 2010), the situation in animal cells appears to be more complicated in that next to glucose (Sautin et al, 2005), assembly can be triggered by amino acids (Stransky & Forgac, 2015), cell maturation (Trombetta et al, 2003), hormones (Voss et al, 2007), and growth factors (Xu et al, 2012). An important role in regulating enzymatic activity in V-ATPase is played by the single-copy subunit H, a 54-kDa two-domain polypeptide found at the interface of V1 and Vo (Wilkens et al, 2004). Subunit H plays a dual role in enzyme function: While H is required for ATP hydrolysis and proton pumping in holo V-ATPase (Ho et al, 1993), the same subunit functions to inhibit MgATPase activity in membrane-detached yeast V1, possibly in combination with product inhibition by ADP (Parra et al, 2000). The molecular mechanisms by which H functions in silencing MgATPase activity in free V1 are not well understood, however, largely due to the lack of detailed structural information. Here, we present the crystal structure of the autoinhibited V1 sector from S. cerevisiae (ScV1) at 6.2–6.5 Å resolution. The structure shows that regulation by reversible dissociation involves a large movement of the C-terminal domain of H (HCT) from its position in V1Vo to a position at the bottom of the A3B3 hexamer in ScV1. Together with accompanying biochemical data, the structure provides a mechanism of activity silencing in the membrane-detached ScV1. Results Crystallographic investigations of the autoinhibited ScV1 While subunit C is released into the cytoplasm during reversible enzyme disassembly (Kane, 1995) (Fig 1C), variable but typically substoichiometric levels of C have been seen to co-purify with ScV1 (Zhang et al, 2003; Diab et al, 2009; Hildenbrand et al, 2010) (see also Fig 5E below). To ensure a homogeneous preparation for crystallogenesis, ScV1 was therefore purified from a yeast strain deleted for the C subunit (Fig EV1A). Initial crystallization screening identified several conditions that resulted in small needle-shaped crystals. Crystal size and diffraction quality were gradually improved by refining a subset of the initial conditions together with additive screening. Crystals used for collecting initial datasets were obtained in 100 mM HEPES, pH 7.5, 150 mM ammonium sulfate, 12.5 mM magnesium or strontium chloride, and 9.5% PEG 8000 using vapor diffusion or a microfluidic device. A 7 Å resolution dataset collected from one ScV1 crystal was used to start the structure determination by molecular replacement (MR) (Table 1). Since there is no crystal structure available for the eukaryotic V1-ATPase, we employed the structure of the nucleotide-free A3B3 catalytic hexamer from the E. hirae sodium pumping V-type ATPase (EhA3B3) (Arai et al, 2013) for MR. The primary structures of E. hirae and yeast A and B subunits are highly similar (48 and 54% identity, respectively), indicating that the tertiary structures of the bacterial and eukaryotic catalytic subunits are conserved and that the bacterial A3B3 catalytic hexamer represents a suitable MR search model for solving the structure of the eukaryotic V1. The MR with EhA3B3 revealed the presence of two ScV1 sectors in the asymmetric unit (ASU) that were related by twofold non-crystallographic symmetry (NCS) (Appendix Fig S1). The density-modified and NCS-averaged MR map revealed electron density not only for A3B3, but also for ScV1 subunits D,E,G,H that were either not present in the bacterial complex (E,G,H) or not part of the MR search model (D) (Fig EV1B). Iterative manual placement of available crystal structures for subunits D,E,G, and H followed by refinement allowed for building of a largely complete model of the ScV1 sector except for subunit F, the base of subunit D and the N-termini of EG2 and EG3 that were not modeled due to insufficient quality of the corresponding electron density. Crystallization conditions were then further refined (8.25% PEG 8000, 250 mM (NH4)2SO4, 100 mM HEPES, pH 7.5, 50 mM SrCl2), resulting in slightly larger crystals that diffracted X-rays anisotropically to ~6.2 Å (Table 1). Diffraction data from two crystals were merged and subjected to elliptic truncation with resolution limits of 6.5, 6.2, and 6.7 Å (Appendix Table S1). This higher resolution (hereafter referred to 6.2–6.5 Å) dataset was used in MR employing the 7 Å structure as search model. The resulting electron density was of higher quality compared to the 7 Å data and allowed for modeling of subunit F, the base of subunit D and extension of the N-termini of peripheral stalks EG2 and EG3 (Appendix Table S2). A stereo representation of the final electron density map is shown in Appendix Fig S2. Note that due to the limited resolution of the diffraction data, all polypeptides used for modeling were truncated at the Cβ-position. The more complete model obtained from the 6.2 to 6.5 Å dataset will be discussed in detail below. Click here to expand this figure. Figure EV1. Purification of ScV1 and density-modified MR map Purification of ScV1. The ˜600-kDa ScV1 (A3B3DE3FG3H) elutes as a single symmetrical peak from the gel filtration column. The boxed area was pooled and concentrated for crystallogenesis of ScV1. Inset: Coomassie blue-stained SDS–PAGE of the pooled and concentrated fractions. NCS-averaged and density-modified MR map contoured at 1.4 σ showing the MR search model (EhA3B3, pink and green; 3vr5). It can be seen that some regions of the MR search model are offset from the corresponding α-helical electron density (see arrow), indicating a slight difference in conformation between catalytic hexamers of ScV1 and EhV1. Also note the extra density extending beyond the search model (labeled as EG1, D, HNT, HCT). The α-helical HEAT repeats of HNT are clearly defined in the map. Download figure Download PowerPoint Table 1. Data collection and refinement statistics (molecular replacement) ScV1 Mg ScV1 Sraa Two crystals. Data collection Space group C 1 2 1 C 1 2 1 Cell dimensions a, b, c (Å) 468.48, 159.74, 245.04 468.02, 159.65, 248.27 α, β, γ (°) 90.00, 113.88, 90.00 90.00, 113.75, 90.00 Resolution (Å) 40.1–7.0 (7.249–7.0)bb Values in parentheses are for highest resolution shell. 39.72–6.211 (6.432–6.211) R merge 0.2558 (2.076) 0.2321 (3.223) I/σI 7.82 (1.41) 12.01 (0.88)cc I/σI in the highest resolution shell after elliptical truncation = 1.51. Completeness (%) 99.0 (94.0) 88.0dd See Appendix Table S1. Redundancy 8.5 (8.4) 11.1 (11.5) CC 1/2 0.988 (0.366) 0.983 (0.377) CC* 0.997 (0.732) 0.996 (0.740) Refinement Resolution (Å) 40.1–7.0 39.72–6.211 No. reflections 26,087 33,464 Rwork/Rfree 26.04/30.9 25.45/30.18 No. atoms Protein 44,760 47,363 Ligand/ion 0 0 Water 0 0 B-factors Protein 291 320 Rms deviations Bond lengths (Å) 0.001 0.002 Bond angles (°) 0.42 0.62 a Two crystals. b Values in parentheses are for highest resolution shell. c I/σI in the highest resolution shell after elliptical truncation = 1.51. d See Appendix Table S1. Overall structure of ScV1 The structure presented here represents the highest resolution information available (to our knowledge) for the autoinhibited ScV1 sector and reveals the conformational changes associated with activity silencing and the loss of binding partners at the membrane. ScV1 can be divided into an A3B3DF catalytic core, three EG heterodimers that serve as peripheral stator stalks (hereafter referred to as peripheral stalks EG1-3) and subunit H, which is unique to the eukaryotic enzyme (Fig 2A). The A3B3DF catalytic core is highly conserved between enzymes from prokaryotes to human and contains the catalytic and non-catalytic interfaces between alternating A and B subunits arranged as a hexamer around a central cavity containing the central rotor composed of subunits D and F (see section “Catalytic hexamer and rotary shaft” below). Each of the three peripheral stalks is bound via its C-terminal domain (ECTGCT) to the N-terminal β-barrel domain of the corresponding B subunit and crosses a non-catalytic A/B interface on its way toward the base of the hexamer (Fig 2B). However, while the stalks' C-terminal domains are largely invariant, their N-termini (ENTGNT) are in different conformations (Fig 2C and D and Appendix Fig S3A). Previously, we have solved the structure of the isolated peripheral stalk heterodimer bound to the C subunit head domain (EGChead, representative of EG3) in two conformations (Oot et al, 2012a). In that study, we provided evidence that the EG heterodimers contain two hinges and a partially disordered “bulge” region in subunit G that provide flexibility and that we speculated would play an important role in the mechanism of reversible disassembly (Appendix Fig S3A and B). In peripheral stalks EG1 and EG2, this segment of G is resolved as continuous tubular density, consistent with α-helical structure. In EG3, however, the density is patchy and flattened, indicating presence of the “bulge” structure as seen in isolated EGChead (Fig 2D and Appendix Fig S3C). Peripheral stalk EG3 is bound to the B subunit of a closed A/B pair and its N-terminal domain is bent inwards, wrapping around the C-terminus of the adjacent catalytic A subunit (ACT) (Fig 2D–F and Movie EV1; see also section “Comparison of autoinhibited ScV1 and ScV1Vo” below). This suggests that the bulge in subunit G may be present or absent depending upon binding partners and/or nucleotide bound state of the enzyme. Figure 2. Overall structure of the autoinhibited V1 Structure of autoinhibited ScV1. The catalytic A (pink) and non-catalytic B (green) subunits are arranged as a heterohexamer around the central rotor subunits D (red) and F (cyan). The three peripheral stalks (EG heterodimers 1–3, blue and orange, respectively) are bound at the periphery of the B subunits. The inhibitory H subunit (yellow) is unique to the eukaryotic V-ATPase. Unless otherwise stated, the coloring scheme used here for the V1 subunits will be used from here on. Interaction of the peripheral stalks with the catalytic hexamer. Each peripheral stalk crosses a non-catalytic A/B interface on its way from the top of the V1 to the base of the catalytic core. Representative view of the interaction is shown for EG3. Interaction of peripheral stalks EG1 and 2 with the catalytic hexamer. The peripheral stalks are bound to the top of the B subunits via their C-termini (boxed). The electron density (contoured at 1.2σ) for the C-terminal domains of EG1 and EG2 is shown in the panels to the left and right. The structure of autoinhibited ScV1 rotated 90° around the vertical from the view shown in panel (A) to highlight peripheral stalks EG2 and EG3. Note the bent appearance of EG3 compared to EG2 along with the presence of a partially unstructured region known as the “bulge” in subunit G (indicated with an arrow). The box shows electron density (contoured at 1.2 σ) for the EG3 N-termini contacting the C-terminal domain of the A subunit of a closed catalytic site (see asterisk). The horizontal lines on the structure indicate the sections shown in panels (E) (upper two lines) and (F) (lower two lines). Section through the ScV1 electron density map showing interaction of the A3B3 catalytic hexamer with the peripheral stalks (EG1–3). View is rotated 90° from the view in (A), looking up the central rotor. The A/B pairs forming the three catalytic sites are designated (AB)1-3 according to which of the three peripheral stalks is bound to each B subunit. For example, (AB)1 has peripheral stalk EG1 bound to its B subunit. The black arrowheads indicate the non-catalytic A/B interfaces. Section through the ScV1 electron density map at the level of the N-termini of the peripheral stalks. Note the change in position of the N-termini of the peripheral stalks, which have each crossed a non-catalytic interface (black arrowheads in E) and are now in proximity to the C-termini of the catalytic A subunits (ACT) of the adjacent A/B pairs. While EG1 and EG2 are now near ACT from (AB)3 and (AB)1, respectively (see double headed arrows), only the bent EG3 peripheral stalk is close enough to contact the corresponding ACT from (AB)2 (see asterisk). Download figure Download PowerPoint The regulatory H subunit can be seen bound to peripheral stalk EG1 via its N-terminal domain (HNT) while its C-terminus (HCT) is bound at the bottom of the catalytic hexamer near the rotary D subunit (Fig 2A and C). The interactions involving H are of particular interest, as this subunit has been shown to be essential for silencing the ATPase activity of the isolated V1 sector (see “Subunit H inhibitory interactions” below). While the interactions between the peripheral stalks and aNT and C are lost in isolated V1, their interactions with the hexamer and the inhibitory subunit H are preserved. The high affinity of the peripheral stalks for the catalytic hexamer and H, combined with the low affinity for the membrane, results in a V1 sector that can rapidly dissociate from the Vo in response to cellular signals, while keeping its inhibitory subunit bound via one of the peripheral stalks. Catalytic hexamer and rotary shaft As mentioned above, the catalytic core (A3B3DF) of V-type rotary ATPases is highly conserved, with primary sequence identities between bacterial, yeast, and mammalian A and B subunits of ~50–80%, respectively (Muench et al, 2011). Not surprisingly therefore, the structure of the ScV1 catalytic core is overall very similar to its bacterial counterpart from E. hirae (EhV1) with an rmsd of 2.3 Å. As in EhV1, the ScV1 catalytic A and non-catalytic B subunits are arranged as a heterohexamer with their N- and C-termini found distal and proximal to the membrane, respectively (Fig 3A). The N-termini of both subunits are folded in a β-barrel, which together form a contiguous β-structure along the top of the molecule (Fig 3A). Alignment of the A subunits by their N-terminal β-barrels illustrates that their C-terminal domains are in different conformations (Fig 3B), consistent with autoinhibited ScV1 containing one open and two closed catalytic sites as is evident in side views of the three A/B pairs (Fig 3C). From here on, the three catalytic sites will be designated (AB)1-3, depending on which of the peripheral stalks EG1-3 is bound to its B subunit, and following this nomenclature, the open catalytic site is (AB)1 and the two closed ones (AB)2 and (AB)3 (Figs 3C and 2E and F). Note that of the two closed catalytic sites, (AB)2 appears more closed than (AB)3 (Fig 3B and C). The presence of two non-equivalently closed sites has been also observed in the EhV1 (Arai et al, 2013) and in the cryo-EM maps of holo eukaryotic V-ATPase (Zhao et al, 2015) (see Discussion section below for more detail). Figure 3. Catalytic A/B pairs and comparison of yeast and bacterial A3B3DF The A3B3DF complex shown with the subunits unique to the eukaryotic V1 removed, highlighting the core conserved catalytic complex labeled as “catalytic core”. The three peripheral stalks and inhibitory subunit H are found only in the eukaryotic V1, whereas A3B3DF represents the full complement of subunits in the prokaryotic V1 complex. Notably, the prokaryotic V1 is an active ATP hydrolase (Arai et al, 2009). The orientation of the A and B subunits with respect to the membrane is indicated by the arrow on the left. Conformational differences in the catalytic A subunits. The catalytic A subunits are shown aligned by their N-terminal β-barrels, highlighting the change in conformation of C-terminal α-helical bundles associated with the closed ((AB)2 and (AB)3; dark and light gray, respectively) and open ((AB)1; magenta) catalytic sites. Movement of this domain in response to nucleotide occupancy is thought to drive rotation of the central rotor during catalysis. ScV1's catalytic hexamer contains one open and two closed catalytic sites. Side view of the three catalytic hexamer A/B pairs (shown in surface representation). Note that the two closed pairs (AB)2 and 3 are non-equivalently closed. Alignment of the A3B3DF from ScV1 and nucleotide-free EhV1 by the catalytic hexamers. The A3B3DF from ScV1 is shown in the same color scheme as in Fig 2, EhV1 is in blue. The alignment shows the difference in position of the central rotor subunit, D. Note that the C-terminus of subunit D (DCT) is longer in eukaryotic V-ATPase (see electron density contoured at 1.2 σ in the box to the right). ScV1 and EhV1 are halted in different rotational positions. Alignment of the A3B3DF of ScV1 and nucleotide-free EhV1 (blue, with dashed lines drawn through the A subunits) by the central stalk subunit D, reveals that the two catalytic cores are halted in different rotational positions. The autoinhibited ScV1 requires 40° rotation to overlay its catalytic hexamer with that of the EhV1. Comparison of the D subunits of ScV1 (red) and nucleotide-free EhV1 (blue). HCT from ScV1 is shown in yellow, and subunit F from EhV1 is shown in dark blue. Download figure Download PowerPoint The central rotor subunit D is an elongated coiled-coil folded as a hairpin, with the N-terminus (DNT) located within and the longer C-terminus (DCT) passing entirely through the A3B3 hexamer (Fig 3A). The ~90-Å-long DNT helix penetrates the hexamer up to the level of the phosphate binding or P-loops that are involved in nucleotide binding (Fig 3A). The ~143–Å-long DCT protrudes from the top of the hexamer by ~10 Å, a feature unique to the eukaryotic enzyme (highlighted by the box in Fig 3D). Subunit F is bound to D below the catalytic hexamer via a conserved C-terminal α-helix (FCT; Fig 3A) in a manner similar to the prokaryotic enzyme (Arai et al, 2013). While density for the FCT α-helix was observed in both of the ScV1 structures solved, only the higher resolution structure allowed modeling of the F subunit N-terminal domain, suggesting some flexibility or mobility of the subunit. Comparing the crystal structures of yeast (ScV1) and bacterial (EhV1) catalytic cores reveals considerable differences regarding the position of the D subunit (Fig 3D). Aligning the structure of autoinhibited ScV1 to available structures of nucleotide-free and AMP-PNP-bound EhV1 (Arai et al, 2013) using the D subunit as reference illustrates that the eukaryotic V1 is halted in a different rotational position (Fig 3E). The largest angular difference observed (40°) was to nucleotide-free EhV1, a noteworthy observation as nucleotide-free EhA3B3 was used as MR search model to solve the structure of ScV1. Besides the difference in rotational position of the D subunit in ScV1 compared to EhV1, the base of the central rotor is bent in the direction of the interaction between the FCT and a B subunit from the hexamer in E. hirae, whereas in ScV1, the base of the central rotor appears to be more straight and shifted toward the C-terminal domain of the H subunit (Fig 3F). Interestingly, a more straight central rotor is also seen in the ADP-bound catalytic core from the T. thermophilus A/V-ATPase (TtV1; Appendix Fig S4) (Nagamatsu et al, 2013), suggesting that the conformation of the DF subcomplex may be influenced by the nucleotide occupancy of the two closed catalytic sites of the complex. Subunit H inhibitory interactions Subunit H was the first component of eukaryotic V-ATPase whose structure was solved by X-ray crystallography (Sagermann et al, 2001). Subunit H is a two-domain polypeptide with a larger N-terminal domain composed of α-helical HEAT repeats (HNT, residues 1–351) connected to a smaller globular α-helical C-terminal domain (HCT, 356–478) by a flexible linker (Fig 4A). While the NCS-averaged and density-modified MR map showed clear electron density for HNT and HCT (Fig EV1B), the two domains of H had to be modeled separately because of the considerable conformational change required for HCT to assume its inhibitory position in ScV1 (discussed in “Comparison of autoinhibited ScV1 and ScV1Vo” below). The electron density for subunit H is of particularly high quality, showing HNT bound to peripheral stalk EG1 and HCT in contact with the base of the catalytic core (Fig 4A). The interaction between HNT and EG1 is largely mediated by two α-helical segments in HNT (α-14 and α-17, comprising residues 237–255 and 284–296) and a short α-helical stretch in subunit E (residues 26–44) (Fig 4B). The contacts between HCT and the catalytic core are mediated by a loop in HCT (residues 408–414) and two short segments in the C-terminal domain of the B subunit of the open catalytic site (AB)1 (residues around Ile427 and Glu471) together with two α-helical turns in DNT (residues 38–45) (Fig 4C). During catalysis, conformational changes in the catalytic hexamer (Fig 3B and C) drive rotation of the central DF rotor (Movie EV2). As the D subunit forms part of the rotor, the site on DNT for the HCT interaction would only be available for binding in this rotational position (Fig 4D). Further, the site on the B subunit for the HCT interaction is only exposed and available for binding in the open conformation of the (AB)1 pair (Movie EV2). From its inhibitory interactions, i" @default.
• W2433610117 created "2016-06-24" @default.
• W2433610117 creator A5001352482 @default.
• W2433610117 creator A5018158537 @default.
• W2433610117 creator A5043740049 @default.
• W2433610117 creator A5086261936 @default.
• W2433610117 date "2016-06-13" @default.
• W2433610117 modified "2023-09-24" @default.
• W2433610117 title "Crystal structure of yeast V ₁ ‐ <scp>ATP</scp> ase in the autoinhibited state" @default.
• W2433610117 cites W1480846298 @default.
• W2433610117 cites W1508637318 @default.
• W2433610117 cites W1551014578 @default.
• W2433610117 cites W1581489141 @default.
• W2433610117 cites W1582076669 @default.
• W2433610117 cites W1625553880 @default.
• W2433610117 cites W1937619740 @default.
• W2433610117 cites W1963903089 @default.
• W2433610117 cites W1968028255 @default.
• W2433610117 cites W1974459427 @default.
• W2433610117 cites W1981283275 @default.
• W2433610117 cites W1981552086 @default.
• W2433610117 cites W1985908916 @default.
• W2433610117 cites W1986246554 @default.
• W2433610117 cites W1986919229 @default.
• W2433610117 cites W1992334712 @default.
• W2433610117 cites W2002801564 @default.
• W2433610117 cites W2009010596 @default.
• W2433610117 cites W2010506265 @default.
• W2433610117 cites W2015808181 @default.
• W2433610117 cites W2021138450 @default.
• W2433610117 cites W2033106369 @default.
• W2433610117 cites W2037565953 @default.
• W2433610117 cites W2039037981 @default.
• W2433610117 cites W2051035135 @default.
• W2433610117 cites W2054456492 @default.
• W2433610117 cites W2056897039 @default.
• W2433610117 cites W2057698787 @default.
• W2433610117 cites W2058876049 @default.
• W2433610117 cites W2061495078 @default.
• W2433610117 cites W2061680908 @default.
• W2433610117 cites W2062562477 @default.
• W2433610117 cites W2064863056 @default.
• W2433610117 cites W2069146114 @default.
• W2433610117 cites W2071320577 @default.
• W2433610117 cites W2077436141 @default.
• W2433610117 cites W2080167396 @default.
• W2433610117 cites W2082377632 @default.
• W2433610117 cites W2082662845 @default.
• W2433610117 cites W2090092599 @default.
• W2433610117 cites W2093961415 @default.
• W2433610117 cites W2116659929 @default.
• W2433610117 cites W2121990753 @default.
• W2433610117 cites W2122023595 @default.
• W2433610117 cites W2131686869 @default.
• W2433610117 cites W2138806334 @default.
• W2433610117 cites W2144701578 @default.
• W2433610117 cites W2150302037 @default.
• W2433610117 cites W2154915448 @default.
• W2433610117 cites W2155216827 @default.
• W2433610117 cites W2177406048 @default.
• W2433610117 cites W2179980862 @default.
• W2433610117 cites W2252527437 @default.
• W2433610117 cites W2258123482 @default.
• W2433610117 doi "https://doi.org/10.15252/embj.201593447" @default.
• W2433610117 hasPubMedCentralId "https://www.ncbi.nlm.nih.gov/pmc/articles/4969575" @default.
• W2433610117 hasPubMedId "https://pubmed.ncbi.nlm.nih.gov/27295975" @default.
• W2433610117 hasPublicationYear "2016" @default.
• W2433610117 type Work @default.
• W2433610117 sameAs 2433610117 @default.
• W2433610117 citedByCount "39" @default.
• W2433610117 countsByYear W24336101172016 @default.
• W2433610117 countsByYear W24336101172017 @default.
• W2433610117 countsByYear W24336101172018 @default.
• W2433610117 countsByYear W24336101172019 @default.
• W2433610117 countsByYear W24336101172020 @default.
• W2433610117 countsByYear W24336101172021 @default.
• W2433610117 countsByYear W24336101172022 @default.
• W2433610117 countsByYear W24336101172023 @default.
• W2433610117 crossrefType "journal-article" @default.
• W2433610117 hasAuthorship W2433610117A5001352482 @default.
• W2433610117 hasAuthorship W2433610117A5018158537 @default.
• W2433610117 hasAuthorship W2433610117A5043740049 @default.
• W2433610117 hasAuthorship W2433610117A5086261936 @default.
• W2433610117 hasBestOaLocation W24336101172 @default.
• W2433610117 hasConcept C153911025 @default.
• W2433610117 hasConcept C2777576037 @default.
• W2433610117 hasConcept C2779222958 @default.
• W2433610117 hasConcept C2910135453 @default.
• W2433610117 hasConcept C55493867 @default.
• W2433610117 hasConcept C86803240 @default.
• W2433610117 hasConcept C95444343 @default.
• W2433610117 hasConceptScore W2433610117C153911025 @default.
• W2433610117 hasConceptScore W2433610117C2777576037 @default.
• W2433610117 hasConceptScore W2433610117C2779222958 @default.
• W2433610117 hasConceptScore W2433610117C2910135453 @default.
• W2433610117 hasConceptScore W2433610117C55493867 @default.
• W2433610117 hasConceptScore W2433610117C86803240 @default.
• W2433610117 hasConceptScore W2433610117C95444343 @default.

• next