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author:("trinity, John")
1.  Structure of the Vacuolar H+-ATPase Rotary Motor Reveals New Mechanistic Insights 
Structure(London, England:1993)  2015;23(3):461-471.
Vacuolar H+-ATPases are multisubunit complexes that operate with rotary mechanics and are essential for membrane proton transport throughout eukaryotes. Here we report a ∼1 nm resolution reconstruction of a V-ATPase in a different conformational state from that previously reported for a lower-resolution yeast model. The stator network of the V-ATPase (and by implication that of other rotary ATPases) does not change conformation in different catalytic states, and hence must be relatively rigid. We also demonstrate that a conserved bearing in the catalytic domain is electrostatic, contributing to the extraordinarily high efficiency of rotary ATPases. Analysis of the rotor axle/membrane pump interface suggests how rotary ATPases accommodate different c ring stoichiometries while maintaining high efficiency. The model provides evidence for a half channel in the proton pump, supporting theoretical models of ion translocation. Our refined model therefore provides new insights into the structure and mechanics of the V-ATPases.
Graphical Abstract
•Subnanometer V-ATPase EM structure gives new insights into mechanism•Comparison of two distinct catalytic states in a complete rotary ATPase•Describes a conserved electrostatic bearing that supports high motor efficiency•Proposes how different c ring stoichiometries are accommodated
Rawson et al. solve a high-resolution structure of vacuolar ATPase. The complex rests in a catalytic state different from those previously reported. The work gives new insights into the organization, mechanism, and the basis for functional properties such as high thermodynamic efficiency.
PMCID: PMC4353692  PMID: 25661654
2.  PA1b Inhibitor Binding to Subunits c and e of the Vacuolar ATPase Reveals Its Insecticidal Mechanism* 
The Journal of Biological Chemistry  2014;289(23):16399-16408.
Background: Pea albumin 1b (PA1b) is a potent and selective inhibitor of insect vacuolar ATPase, but its mechanism is poorly understood.
Results: PA1b binds the extracellular surface of the c ring and contacts subunit e.
Conclusion: PA1b inhibits V-ATPase by blocking transition of its rotor past subunit e.
Significance: This reveals insights into the mechanics and mode of inhibition of the V-ATPase.
The vacuolar ATPase (V-ATPase) is a 1MDa transmembrane proton pump that operates via a rotary mechanism fuelled by ATP. Essential for eukaryotic cell homeostasis, it plays central roles in bone remodeling and tumor invasiveness, making it a key therapeutic target. Its importance in arthropod physiology also makes it a promising pesticide target. The major challenge in designing lead compounds against the V-ATPase is its ubiquitous nature, such that any therapeutic must be capable of targeting particular isoforms. Here, we have characterized the binding site on the V-ATPase of pea albumin 1b (PA1b), a small cystine knot protein that shows exquisitely selective inhibition of insect V-ATPases. Electron microscopy shows that PA1b binding occurs across a range of equivalent sites on the c ring of the membrane domain. In the presence of Mg·ATP, PA1b localizes to a single site, distant from subunit a, which is predicted to be the interface for other inhibitors. Photoaffinity labeling studies show radiolabeling of subunits c and e. In addition, weevil resistance to PA1b is correlated with bafilomycin resistance, caused by mutation of subunit c. The data indicate a binding site to which both subunits c and e contribute and inhibition that involves locking the c ring rotor to a static subunit e and not subunit a. This has implications for understanding the V-ATPase mechanism and that of inhibitors with therapeutic or pesticidal potential. It also provides the first evidence for the position of subunit e within the complex.
PMCID: PMC4047407  PMID: 24795045
ATPase; Drug Action; Electron Microscopy (EM); Membrane Protein; Vacuolar ATPase; PA1b
3.  1H, 15N and 13C backbone chemical shift assignment of titin domains A59–A60 and A60 alone 
Biomolecular Nmr Assignments  2014;8(2):429-433.
The giant protein titin is the third most abundant protein of vertebrate striated muscle. The titin molecule is >1 μm long and spans half the sarcomere, from the Z-disk to the M-line, and has important roles in sarcomere assembly, elasticity and intracellular signaling. In the A-band of the sarcomere titin is attached to the thick filaments and mainly consists immunoglobulin-like and fibronectin type III-like domains. These are mostly arranged in long-range patterns or ‘super-repeats’. The large super-repeats each contain 11 domains and are repeated 11 times, thus forming nearly half the titin molecule. Through interactions with myosin and C-protein, they are involved in thick filament assembly. The importance of titin in muscle assembly is highlighted by the effect of mutations in the A-band portion, which are the commonest cause of dilated cardiomyopathy, affecting ~1 in 250 (Herman et al. in N Engl J Med 366:619–628, 2012). Here we report backbone 15N, 13C and 1H chemical shift and 13Cβ assignments for the A59–A60 domain tandem from the titin A59–A69 large super-repeat, completed using triple resonance NMR. Since, some regions of the backbone remained unassigned in A60 domain of the complete A59–A60 tandem, a construct containing a single A60 domain, A60sd, was also studied using the same methods. Considerably improved assignment coverage was achieved using A60sd due to its lower mass and improved molecular tumbling rate; these assignments also allowed the analysis of inter-domain interactions using chemical shift mapping against A59–A60.
PMCID: PMC4145206  PMID: 24469996
Muscle protein; Titin A-band; Large super-repeat unit; Fibronectin type III domain tandem
4.  Subunit Positioning and Stator Filament Stiffness in Regulation and Power Transmission in the V1 Motor of the Manduca sexta V-ATPase☆ 
Journal of Molecular Biology  2014;426(2):286-300.
The vacuolar H+-ATPase (V-ATPase) is an ATP-driven proton pump essential to the function of eukaryotic cells. Its cytoplasmic V1 domain is an ATPase, normally coupled to membrane-bound proton pump Vo via a rotary mechanism. How these asymmetric motors are coupled remains poorly understood. Low energy status can trigger release of V1 from the membrane and curtail ATP hydrolysis. To investigate the molecular basis for these processes, we have carried out cryo-electron microscopy three-dimensional reconstruction of deactivated V1 from Manduca sexta. In the resulting model, three peripheral stalks that are parts of the mechanical stator of the V-ATPase are clearly resolved as unsupported filaments in the same conformations as in the holoenzyme. They are likely therefore to have inherent stiffness consistent with a role as flexible rods in buffering elastic power transmission between the domains of the V-ATPase. Inactivated V1 adopted a homogeneous resting state with one open active site adjacent to the stator filament normally linked to the H subunit. Although present at 1:1 stoichiometry with V1, both recombinant subunit C reconstituted with V1 and its endogenous subunit H were poorly resolved in three-dimensional reconstructions, suggesting structural heterogeneity in the region at the base of V1 that could indicate positional variability. If the position of H can vary, existing mechanistic models of deactivation in which it binds to and locks the axle of the V-ATPase rotary motor would need to be re-evaluated.
Graphical abstract
•Dissociation of vacuolar H+-ATPase domains deactivates its V1 motor.•V1 has one “open” catalytic site linked to the stator filament bound by subunit H.•Movement of subunit H to prevent rotary catalysis is possible.•Three stator filaments project from deactivated V1, indicating inherent stiffness.•This work gives new insight into energetic coupling and control in V-ATPases.
PMCID: PMC3899036  PMID: 24075871
3D, three dimensional; EM, electron microscopy; vacuolar membrane; H+-ATPase; cryo-electron microscopy
5.  Flexibility within the Rotor and Stators of the Vacuolar H+-ATPase  
PLoS ONE  2013;8(12):e82207.
The V-ATPase is a membrane-bound protein complex which pumps protons across the membrane to generate a large proton motive force through the coupling of an ATP-driven 3-stroke rotary motor (V1) to a multistroke proton pump (Vo). This is done with near 100% efficiency, which is achieved in part by flexibility within the central rotor axle and stator connections, allowing the system to flex to minimise the free energy loss of conformational changes during catalysis. We have used electron microscopy to reveal distinctive bending along the V-ATPase complex, leading to angular displacement of the V1 domain relative to the Vo domain to a maximum of ~30°. This has been complemented by elastic network normal mode analysis that shows both flexing and twisting with the compliance being located in the rotor axle, stator filaments, or both. This study provides direct evidence of flexibility within the V-ATPase and by implication in related rotary ATPases, a feature predicted to be important for regulation and their high energetic efficiencies.
PMCID: PMC3846802  PMID: 24312643
6.  Making Muscle Elastic: The Structural Basis of Myomesin Stretching 
PLoS Biology  2012;10(2):e1001264.
The muscle M-band protein myomesin comprises a 36-nm long filament made of repetitive immunoglobulin–helix modules that can stretch to 2.5-fold this length, demonstrating substantial molecular elasticity.
Skeletal and cardiac muscles are remarkable biological machines that support and move our bodies and power the rhythmic work of our lungs and hearts. As well as producing active contractile force, muscles are also passively elastic, which is essential to their performance. The origins of both active contractile and passive elastic forces can be traced to the individual proteins that make up the highly ordered structure of muscle. In this Primer, we describe the organization of sarcomeres—the structural units that produce contraction—and the nature of the proteins that make muscle elastic. In particular, we focus on an elastic protein called myomesin, whose novel modular architecture helps explain elasticity.
PMCID: PMC3279349  PMID: 22347814
7.  An approach to automated acquisition of cryoEM images from lacey carbon grids 
Journal of structural biology  2010;172(3):395-399.
An approach to automated acquisition of cryoEM image data from lacey carbon grids using the Leginon program is described. Automated liquid nitrogen top up of the specimen holder dewar was used as a step towards full automation, without operator intervention during the course of data collection. During cryoEM studies of actin labelled with myosin V, we have found it necessary to work with lacey grids rather than Quantifoil or C-flat grids due to interaction of myosin V with the support film. Lacey grids have irregular holes of variable shape and size, in contrast to Quantifoil or C-flat grids which have a regular array of similar circular holes on each grid square. Other laboratories also prefer to work with grids with irregular holes for a variety of reasons. Therefore, it was necessary to develop a different strategy from normal Leginon usage for working with lacey grids for targetting holes for image acquisition and suitable areas for focussing prior to image acquisition. This approach was implemented by using the extensible framework provided by Leginon and by developing a new MSI application within that framework which includes a new Leginon node (for a novel method for finding focus targets).
PMCID: PMC3001122  PMID: 20817100
Electronmicroscopy; Data collection; Single-particle reconstruction; High throughput
8.  Roles of Titin in the Structure and Elasticity of the Sarcomere 
The giant protein titin is thought to play major roles in the assembly and function of muscle sarcomeres. Structural details, such as widths of Z- and M-lines and periodicities in the thick filaments, correlate with the substructure in the respective regions of the titin molecule. Sarcomere rest length, its operating range of lengths, and passive elastic properties are also directly controlled by the properties of titin. Here we review some recent titin data and discuss its implications for sarcomere architecture and elasticity.
PMCID: PMC2896707  PMID: 20625501
9.  Preservation of high resolution protein structure by cryo-electron microscopy of vitreous sections 
Ultramicroscopy  2009;110(1):43-47.
We have quantitated the degree of structural preservation in cryo-sections of a vitrified biological specimen. Previous studies have used sections of periodic specimens to assess the resolution present, but preservation before sectioning was not assessed and so the damage due particularly to cutting was not clear. In this study large single crystals of lysozyme were vitrified and from these X-ray diffraction patterns extending to better than 2.1 Å were obtained. The crystals were high pressure frozen in 30% dextran, and cryo-sectioned using a diamond knife. In the best case, preservation to a resolution of 7.9 Å was shown by electron diffraction, the first observation of sub-nanometre structural preservation in a vitreous section.
PMCID: PMC2824107  PMID: 19819624
Cryo-section; Section resolution
10.  3D morphology of the human hepatic ferritin mineral core: New evidence for a subunit structure revealed by single particle analysis of HAADF-STEM images 
Journal of Structural Biology  2009;166(1):22-31.
Ferritin, the major iron storage protein, has dual functions; it sequesters redox activity of intracellular iron and facilitates iron turn-over. Here we present high angle annular dark field (HAADF) images from individual hepatic ferritin cores within tissue sections, these images were obtained using spherical aberration corrected scanning transmission electron microscopy (STEM) under controlled electron fluence. HAADF images of the cores suggest a cubic morphology and a polycrystalline (ferrihydrite) subunit structure that is not evident in equivalent bright field images. By calibrating contrast levels in the HAADF images using quantitative electron energy loss spectroscopy, we have estimated the absolute iron content in any one core, and produced a three dimensional reconstruction of the average core morphology. The core is composed of up to eight subunits, consistent with the eight channels in the protein shell that deliver iron to the central cavity. We find no evidence of a crystallographic orientation relationship between core subunits. Our results confirm that the ferritin protein shell acts as a template for core morphology and within the core, small (∼2 nm), surface-disordered ferrihydrite subunits connect to leave a low density centre and a high surface area that would allow rapid turn-over of iron in biological systems.
PMCID: PMC2832756  PMID: 19116170
Ferritin; Ferritin core subunit structure; Scanning transmission electron microscopy (STEM); Electron fluence; Electron energy loss spectroscopy (EELS)
11.  The structure of M.EcoKI Type I DNA methyltransferase with a DNA mimic antirestriction protein 
Nucleic Acids Research  2008;37(3):762-770.
Type-I DNA restriction–modification (R/M) systems are important agents in limiting the transmission of mobile genetic elements responsible for spreading bacterial resistance to antibiotics. EcoKI, a Type I R/M enzyme from Escherichia coli, acts by methylation- and sequence-specific recognition, leading to either methylation of DNA or translocation and cutting at a random site, often hundreds of base pairs away. Consisting of one specificity subunit, two modification subunits, and two DNA translocase/endonuclease subunits, EcoKI is inhibited by the T7 phage antirestriction protein ocr, a DNA mimic. We present a 3D density map generated by negative-stain electron microscopy and single particle analysis of the central core of the restriction complex, the M.EcoKI M2S1 methyltransferase, bound to ocr. We also present complete atomic models of M.EcoKI in complex with ocr and its cognate DNA giving a clear picture of the overall clamp-like operation of the enzyme. The model is consistent with a large body of experimental data on EcoKI published over 40 years.
PMCID: PMC2647291  PMID: 19074193
12.  Myosin VI: cellular functions and motor properties. 
Myosin VI has been localized in membrane ruffles at the leading edge of cells, at the trans-Golgi network compartment of the Golgi complex and in clathrin-coated pits or vesicles, indicating that it functions in a wide variety of intracellular processes. Myosin VI moves along actin filaments towards their minus end, which is the opposite direction to all of the other myosins so far studied (to our knowledge), and is therefore thought to have unique properties and functions. To investigate the cellular roles of myosin VI, we identified various myosin VI binding partners and are currently characterizing their interactions within the cell. As an alternative approach, we have expressed and purified full-length myosin VI and studied its in vitro properties. Previous studies assumed that myosin VI was a dimer, but our biochemical, biophysical and electron microscopic studies reveal that myosin VI can exist as a stable monomer. We observed, using an optical tweezers force transducer, that monomeric myosin VI is a non-processive motor which, despite a relatively short lever arm, generates a large working stroke of 18 nm. Whether monomer and/or dimer forms of myosin VI exist in cells and their possible functions will be discussed.
PMCID: PMC1693462  PMID: 15647169
13.  The prepower stroke conformation of myosin V 
The Journal of Cell Biology  2002;159(6):983-991.
eW have used electron microscopy and single-particle image processing to study head conformation in myosin V molecules. We find that in the presence of ATP, many heads have a sharply angled conformation that is rare in its absence. The sharply angled conformation is similar to a myosin II atomic structure proposed to mimic the prepower stroke state. The leading head in molecules attached to actin by both heads has a similar conformation, but is also sharply angled in a second plane by tethering through the trail head. The lead head lever joins the motor domain ∼5 nm axially from where it joins the trail motor. These positions locate the converter subdomain and show the lead motor is in the prepower stroke conformation. Tethering by the trail head places the lead head motor domain at the correct axial position along the actin for binding, but at the wrong orientation. Attachment is achieved either by bending the lead head lever throughout its length or at the pliant point. The microscopy shows that most of the walking stride is produced by changes in lever angle brought about by converter movement, but is augmented by distortion produced by thermal energy.
PMCID: PMC2173995  PMID: 12499355
titling lever; force; thermal ratchet; mechanism; walking

Results 1-13 (13)