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 cancer1. The hydrophilic V1 portion is known as a rotary motor, in which a central axis DF complex rotates inside a hexagonally arranged catalytic A3B3 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 V1-ATPase from the A3B3 and DF complexes2, 3. Here we report the asymmetric structures of the nucleotide-free (2.8 Å) and nucleotide-bound (3.4 Å) A3B3 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) V1-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 V1-ATPase.
At a glance
- Vacuolar ATPases: rotary proton pumps in physiology and pathophysiology. Nature Rev. Mol. Cell Biol. 8, 917–929 (2007)
- Reconstitution in vitro of the catalytic portion (NtpA3-B3-D-G complex) of Enterococcus hirae V-type Na+-ATPase. Biochem. Biophys. Res. Commun. 390, 698–702 (2009) et al.
- Crystal structure of the central axis DF complex of the prokaryotic V-ATPase. Proc. Natl Acad. Sci. USA 108, 19955–19960 (2011) et al.
- ATP synthesis by rotary catalysis (Nobel Lecture). Angew. Chem. Int. Edn Engl. 37, 2308–2319 (1998)
- Inventing the dynamo machine: the evolution of the F-type and V-type ATPases. Nature Rev. Microbiol. 5, 892–899 (2007) , , &
- Structure at 2.8 Å resolution of F1-ATPase from bovine heart mitochondria. Nature 370, 621–628 (1994) , , &
- Structure of bovine mitochondrial F1-ATPase with nucleotide bound to all three catalytic sites: implications for the mechanism of rotary catalysis. Cell 106, 331–341 (2001) , &
- The structure of bovine F1-ATPase inhibited by ADP and beryllium fluoride. EMBO J. 23, 2734–2744 (2004) , , , &
- Ground state structure of F1-ATPase from bovine heart mitochondria at 1.9 Å resolution. J. Biol. Chem. 282, 14238–14242 (2007) , , &
- Novel features of the rotary catalytic mechanism revealed in the structure of yeast F1 ATPase. EMBO J. 25, 5433–5442 (2006) , , , &
- Asymmetric structure of the yeast F1 ATPase in the absence of bound nucleotides. J. Biol. Chem. 284, 10546–10551 (2009) et al.
- Molecular architecture of the rotary motor in ATP synthase. Science 286, 1700–1705 (1999) , &
- The crystal structure of the nucleotide-free α3β3 subcomplex of F1-ATPase from the thermophilic Bacillus PS3 is a symmetric trimer. Structure 5, 825–836 (1997) et al.
- Structure of the ATP synthase catalytic complex (F1) from Escherichia coli in an autoinhibited conformation. Nature Struct. Mol. Biol. 18, 701–707 (2011) &
- Direct observation of the rotation of F1-ATPase. Nature 386, 299–302 (1997) , , &
- Resolution of distinct rotational substeps by submillisecond kinetic analysis of F1-ATPase. Nature 410, 898–904 (2001) , , , &
- Coupling of rotation and catalysis in F1-ATPase revealed by single-molecule imaging and manipulation. Cell 130, 309–321 (2007) et al.
- Dodecamer rotor ring defines H+/ATP ratio for ATP synthesis of prokaryotic V-ATPase from Thermus thermophilus. Proc. Natl Acad. Sci. USA 104, 20256–20261 (2007) et al.
- Crystal structure of A3B3 complex of V-ATPase from Thermus thermophilus. EMBO J. 28, 3771–3779 (2009) et al.
- Inter-subunit interaction and quaternary rearrangement defined by the central stalk of prokaryotic V1-ATPase. EMBO Rep. 10, 1228–1234 (2009) , , &
- Rotation scheme of V1-motor is different from that of F1-motor. Proc. Natl Acad. Sci. USA 102, 17929–17933 (2005) et al.
- Na+ binding of V-type Na+-ATPase in Enterococcus hirae. J. Biol. Chem. 275, 13415–13419 (2000) , , &
- Structure of the rotor of the V-Type Na+-ATPase from Enterococcus hirae. Science 308, 654–659 (2005) , , , &
- Ion binding and selectivity of the rotor ring of the Na+-transporting V-ATPase. Proc. Natl Acad. Sci. USA 105, 8607–8612 (2008) et al.
- Structure of the rotor ring modified with N,N-dicyclohexylcarbodiimide of the Na+-transporting vacuolar ATPase. Proc. Natl Acad. Sci. USA 108, 13474–13479 (2011) et al.
- Structure and mechanism of vacuolar Na+-translocating ATPase from Enterococcus hirae. J. Bioenerg. Biomembr. 37, 411–413 (2005) , &
- Interaction and stoichiometry of the peripheral stalk subunits NtpE and NtpF and the N-terminal hydrophilic domain of NtpI of Enterococcus hirae V-ATPase. J. Biol. Chem. 283, 19422–19431 (2008) et al.
- Mass spectrometry of intact V-type ATPases reveals bound lipids and the effects of nucleotide binding. Science 334, 380–385 (2011) et al.
- Site-directed mutagenesis of the yeast V-ATPase A subunit. J. Biol. Chem. 272, 11750–11756 (1997) et al.
- On the mechanism of ATP hydrolysis in F1-ATPase. Biophys. J. 85, 2253–2266 (2003) , &
- Preparation of Escherichia coli cell extract for highly productive cell-free protein expression. J. Struct. Funct. Genomics 5, 63–68 (2004) et al.
- Purification and properties of firefly luciferase. Methods Enzymol. 57, 3–15 (1978) &
- iMOSFLM: a new graphical interface for diffraction-image processing with MOSFLM. Acta Crystallogr. D 67, 271–281 (2011) , , , &
- Collaborative Computational Project, Number 4. The CCP4 suite: programs for protein crystallography. Acta Crystallogr. D 50, 760–763 (1994)
- Molecular replacement with MOLREP. Acta Crystallogr. D 66, 22–25 (2010) &
- XDS. Acta Crystallogr. D 66, 125–132 (2010)
- Phaser crystallographic software. J. Appl. Crystallogr. 40, 658–674 (2007) et al.
- Coot: model-building tools for molecular graphics. Acta Crystallogr. D 60, 2126–2132 (2004) &
- Refinement of macromolecular structures by the maximum-likelihood method. Acta Crystallogr. D 53, 240–255 (1997) , &
- PHENIX: a comprehensive Python-based system for macromolecular structure solution. Acta Crystallogr. D 66, 213–221 (2010) et al.
- Validation of protein models derived from experiment. Curr. Opin. Struct. Biol. 8, 631–639 (1998) , &
- Structure validation by Cα geometry: φ, ψ and Cβ deviation. Proteins 50, 437–450 (2003) et al.
- Video 1: conformational changes of A3B3 complex induced by AMP-PNP:Mg binding (9,919 KB, Download)
- The colouring and viewing are consistent with Fig. 1. The video was generated by morphing between X-ray crystal structures of nucleotide-free (eA3B3) and nucleotide-bound (bA3B3) A3B3 complexes using PyMOL.
- Video 2: conformational changes of nucleotide-free eA3B3 complex induced by DF binding (12,727 KB, Download)
- The colouring and viewing are consistent with Fig. 2. The video was generated by morphing between X-ray crystal structures of nucleotide-free A3B3 (eA3B3) and A3B3DF (eV1) complexes using PyMOL.
- Video 3: conformational changes of nucleotide-bound A3B3 complex induced by DF binding (13,168 KB, Download)
- The colouring and viewing are consistent with Fig. 2. The video was generated by morphing between X-ray crystal structures of nucleotide-bound A3B3 (bA3B3) and A3B3DF (bV1) complexes using PyMOL.
- Video 4: conformational difference of the nucleotide-binding sites of nucleotide-bound V1-ATPase (2,162 KB, Download)
- The colouring and viewing are consistent with Fig. 3. The video was generated by morphing between X-ray crystal structures of the ACBO’ pair (bound form) and ACRBCR pair (tight form) in nucleotide-bound A3B3DF (bV1) complex using PyMOL.
- Supplementary Information (4.6 MB)
This file contains Supplementary Figures 1-17, Supplementary Tables 1-5, a Supplementary Discussion and Supplementary References. This file was replaced on 21 January 2013 as the original file posted online had corrupted.