The transport mechanism of P4 ATPase lipid flippases

2020 ◽  
Vol 477 (19) ◽  
pp. 3769-3790
Author(s):  
Rosa L. López-Marqués ◽  
Pontus Gourdon ◽  
Thomas Günther Pomorski ◽  
Michael Palmgren
Keyword(s):  
Transport Mechanism ◽  
Structural Models ◽  
Lipid Transport ◽  
Specific Lipids ◽  
Specific Expression ◽  
X Ray ◽  
X Ray Crystallography ◽  
Cellular Processes ◽  
Order Of Magnitude ◽  
Intense Debate

P4 ATPase lipid flippases are ATP-driven transporters that translocate specific lipids from the exoplasmic to the cytosolic leaflet of biological membranes, thus establishing a lipid gradient between the two leaflets that is essential for many cellular processes. While substrate specificity, subcellular and tissue-specific expression, and physiological functions have been assigned to a number of these transporters in several organisms, the mechanism of lipid transport has been a topic of intense debate in the field. The recent publication of a series of structural models based on X-ray crystallography and cryo-EM studies has provided the first glimpse into how P4 ATPases have adapted the transport mechanism used by the cation-pumping family members to accommodate a substrate that is at least an order of magnitude larger than cations.

scholarly journals Mechanism of IPPase shown by high resolution Neutron and X-ray crystallography

2014 ◽  
Vol 70 (a1) ◽  
pp. C1211-C1211
Author(s):  
Joseph Ng ◽  
Ronny Hughes ◽  
Michelle Morris ◽  
Leighton Coates ◽  
Matthew Blakeley ◽  
...  
Keyword(s):  
High Resolution ◽  
Active Site ◽  
Inorganic Pyrophosphatase ◽  
Inorganic Pyrophosphate ◽  
X Ray ◽  
X Ray Crystallography ◽  
Cellular Processes ◽  
Crystallographic Structures ◽  
Hydrolysis Of ◽  
Low Energy Conformations

Soluble inorganic pyrophosphatase (IPPase) catalyzes the hydrolysis of inorganic pyrophosphate (PPi) to form orthophosphate (Pi). The action of this enzyme shifts the overall equilibrium in favor of synthesis during a number of ATP-dependent cellular processes such as in the polymerization of nucleic acids, production of coenzymes and proteins and sulfate assimilation pathways. Two Neutron crystallographic (2.10-2.50Å) and five high-resolution X-ray (0.99Å-1.92Å) structures of the archaeal IPPase from Thermococcus thioreducens have been determined under both cryo and room temperatures. The structures determined include the recombinant IPPase bound to Mg+2, Ca+2, Br-, SO2-2 or PO4-2 involving those with non-hydrolyzed and hydrolyzed pyrophosphate complexes. All the crystallographic structures provide snapshots of the active site corresponding to different stages of the hydrolysis of inorganic pyrophosphate. As a result, a structure-based model of IPPase catalysis is devised showing the enzyme's low-energy conformations, hydration states, movements and nucleophile generation within the active site.

scholarly journals Structure and transport mechanism of the sodium/proton antiporter MjNhaP1

eLife ◽  
2014 ◽  
Vol 3 ◽  
Author(s):  
Cristina Paulino ◽  
David Wöhlert ◽  
Ekaterina Kapotova ◽  
Özkan Yildiz ◽  
Werner Kühlbrandt
Keyword(s):  
Transport Mechanism ◽  
Ion Binding ◽  
Ph Homeostasis ◽  
Activity Maximum ◽  
X Ray ◽  
X Ray Crystallography ◽  
Highly Active ◽  
Health And Disease ◽  
Cooperative Transport ◽  
22Na Uptake

Sodium/proton antiporters are essential for sodium and pH homeostasis and play a major role in human health and disease. We determined the structures of the archaeal sodium/proton antiporter MjNhaP1 in two complementary states. The inward-open state was obtained by x-ray crystallography in the presence of sodium at pH 8, where the transporter is highly active. The outward-open state was obtained by electron crystallography without sodium at pH 4, where MjNhaP1 is inactive. Comparison of both structures reveals a 7° tilt of the 6 helix bundle. 22Na+ uptake measurements indicate non-cooperative transport with an activity maximum at pH 7.5. We conclude that binding of a Na+ ion from the outside induces helix movements that close the extracellular cavity, open the cytoplasmic funnel, and result in a ∼5 Å vertical relocation of the ion binding site to release the substrate ion into the cytoplasm.

scholarly journals Probing the Structure of [NiFeSe] Hydrogenase with QM/MM Computations

2020 ◽  
Vol 10 (3) ◽  
pp. 781 ◽  
Author(s):  
Samah Moubarak ◽  
N. Elghobashi-Meinhardt ◽  
Daria Tombolelli ◽  
Maria Andrea Mroginski
Keyword(s):  
Iron Oxidation ◽  
Structural Models ◽  
Oxidation States ◽  
Quantum Mechanical ◽  
Desulfovibrio Vulgaris ◽  
X Ray ◽  
X Ray Crystallography ◽  
Desulfovibrio Vulgaris Hildenborough ◽  
Optimized Geometries ◽  
Good Agreement

The geometry and vibrational behavior of selenocysteine [NiFeSe] hydrogenase isolated from Desulfovibrio vulgaris Hildenborough have been investigated using a hybrid quantum mechanical (QM)/ molecular mechanical (MM) approach. Structural models have been built based on the three conformers identified in the recent crystal structure resolved at 1.3 Å from X-ray crystallography. In the models, a diamagnetic Ni2+ atom was modeled in combination with both Fe2+ and Fe3+ to investigate the effect of iron oxidation on geometry and vibrational frequency of the nonproteic ligands, CO and CN-, coordinated to the Fe atom. Overall, the QM/MM optimized geometries are in good agreement with the experimentally resolved geometries. Analysis of computed vibrational frequencies, in comparison with experimental Fourier-transform infrared (FTIR) frequencies, suggests that a mixture of conformers as well as Fe2+ and Fe3+ oxidation states may be responsible for the acquired vibrational spectra.

scholarly journals Where is crystallography going?

2018 ◽  
Vol 74 (2) ◽  
pp. 152-166 ◽  
Author(s):  
Jonathan M. Grimes ◽  
David R. Hall ◽  
Alun W. Ashton ◽  
Gwyndaf Evans ◽  
Robin L. Owen ◽  
...  
Keyword(s):  
Electron Microscopy ◽  
Synchrotron Radiation ◽  
Electron Diffraction ◽  
Recombinant Proteins ◽  
Free Electron Lasers ◽  
Macromolecular Crystallography ◽  
Signal To Noise ◽  
X Ray ◽  
X Ray Crystallography ◽  
Order Of Magnitude

Macromolecular crystallography (MX) has been a motor for biology for over half a century and this continues apace. A series of revolutions, including the production of recombinant proteins and cryo-crystallography, have meant that MX has repeatedly reinvented itself to dramatically increase its reach. Over the last 30 years synchrotron radiation has nucleated a succession of advances, ranging from detectors to optics and automation. These advances, in turn, open up opportunities. For instance, a further order of magnitude could perhaps be gained in signal to noise for general synchrotron experiments. In addition, X-ray free-electron lasers offer to capture fragments of reciprocal space without radiation damage, and open up the subpicosecond regime of protein dynamics and activity. But electrons have recently stolen the limelight: so is X-ray crystallography in rude health, or will imaging methods, especially single-particle electron microscopy, render it obsolete for the most interesting biology, whilst electron diffraction enables structure determination from even the smallest crystals? We will lay out some information to help you decide.

One Order of Magnitude Increase of Triplet State Lifetime Observed in Deprotonated Form Selenium Substituted Uracil

2021 ◽  
Author(s):  
Peipei Jin ◽  
Xueli Wang ◽  
Haifeng Pan ◽  
Jinquan Chen
Keyword(s):  
Nucleic Acids ◽  
Triplet State ◽  
Deprotonated Form ◽  
X Ray ◽  
X Ray Crystallography ◽  
Wide Range ◽  
Order Of Magnitude ◽  
Medical Therapies ◽  
Magnitude Increase

Selenium nucleic acids possess unique properties and have been demonstrated to have a wide range of applications such as DNA X-ray crystallography and novel medical therapies. Yet, as a heavy...

scholarly journals Structural Models for Cytochrome P450�Mediated Catalysis

2003 ◽  
Vol 3 ◽  
pp. 536-545 ◽  
Author(s):  
David F.V. Lewis
Keyword(s):  
Cytochrome P450 ◽  
Homology Modelling ◽  
Structural Models ◽  
Structural Factors ◽  
Subsequent Formation ◽  
X Ray ◽  
X Ray Crystallography ◽  
Current Review ◽  
Mediated Catalysis ◽  
Structure Calculations

This review focuses on the structural models for cytochrome P450 that are improving our knowledge and understanding of the P450 catalytic cycle, and the way in which substrates bind to the enzyme leading to catalytic conversion and subsequent formation of mono-oxygenated metabolites. Various stages in the P450 reaction cycle have now been investigated using X-ray crystallography and electronic structure calculations, whereas homology modelling of mammalian P450s is currently revealing important aspects of pharmaceutical and other xenobiotic metabolism mediated by P450 involvement. These features are explored in the current review on P450-based catalysis, which emphasises the importance of structural modelling to our understanding of this enzyme's function. In addition, the results of various QSAR analyses on series of chemicals, which are metabolised via P450 enzymes, are presented such that the importance of electronic and other structural factors in explaining variations in rates of metabolism can be appreciated.

Membrane protein crystallography in the era of modern structural biology

2020 ◽  
Vol 48 (6) ◽  
pp. 2505-2524
Author(s):  
Tristan O. C. Kwan ◽  
Danny Axford ◽  
Isabel Moraes
Keyword(s):  
Structural Biology ◽  
Molecular Level ◽  
Protein Structures ◽  
Three Dimensional ◽  
Biological Macromolecules ◽  
X Ray ◽  
X Ray Crystallography ◽  
Cellular Processes ◽  
Biochemical Level

The aim of structural biology has been always the study of biological macromolecules structures and their mechanistic behaviour at molecular level. To achieve its goal, multiple biophysical methods and approaches have become part of the structural biology toolbox. Considered as one of the pillars of structural biology, X-ray crystallography has been the most successful method for solving three-dimensional protein structures at atomic level to date. It is however limited by the success in obtaining well-ordered protein crystals that diffract at high resolution. This is especially true for challenging targets such as membrane proteins (MPs). Understanding structure-function relationships of MPs at the biochemical level is vital for medicine and drug discovery as they play critical roles in many cellular processes. Though difficult, structure determination of MPs by X-ray crystallography has significantly improved in the last two decades, mainly due to many relevant technological and methodological developments. Today, numerous MP crystal structures have been solved, revealing many of their mechanisms of action. Yet the field of structural biology has also been through significant technological breakthroughs in recent years, particularly in the fields of single particle electron microscopy (cryo-EM) and X-ray free electron lasers (XFELs). Here we summarise the most important advancements in the field of MP crystallography and the significance of these developments in the present era of modern structural biology.

scholarly journals Slow thrombin in solution

2005 ◽  
Vol 390 (2) ◽  
Author(s):  
James A. Huntington
Keyword(s):  
Protein Structures ◽  
Structural Models ◽  
Biological Mechanisms ◽  
New Paradigm ◽  
Field Of Study ◽  
X Ray ◽  
X Ray Crystallography ◽  
Exponential Increase ◽  
Biochemical Journal ◽  
Crystallization Conditions

As a tool for understanding biological mechanisms, X-ray crystallography possesses unparalleled power to enlighten, resolve controversy and shift a field of study on to a secure new paradigm. Thanks largely to developments in crystallographic methods, the technique has become accessible to the general biochemist and we have thus witnessed an exponential increase in the number of protein structures deposited every year. It is now commonplace for several structures to be published of the same protein under different crystallization conditions, sometimes resulting in conflicting mechanistic interpretations. Such a controversy has arisen over thrombin's conformational response to Na+ co-ordination, and in this issue of Biochemical Journal, De Filippis and colleagues put the two structural models of thrombin allostery to the test by returning to the techniques of solution biochemistry.

Photosystem II: the engine of life

2003 ◽  
Vol 36 (1) ◽  
pp. 71-89 ◽  
Author(s):  
James Barber
Keyword(s):  
High Resolution ◽  
Photosystem Ii ◽  
Water Oxidation ◽  
Structural Models ◽  
Reaction Centre ◽  
Ps Ii ◽  
X Ray ◽  
X Ray Crystallography ◽  
Redox Active ◽  
Resolution Electron

1. Introduction 712. Electron transfer in PS II 723. (Mn)4cluster and mechanism of water oxidation 734. Organization and structure of the protein subunits 755. Organization of chlorophylls and redox active cofactors 816. Implications arising from the structural models 827. Perspectives 848. Acknowledgements 869. Addendum 8610. References 87Photosystem II (PS II) is a multisubunit membrane protein complex, which uses light energy to oxidize water and reduce plastoquinone. High-resolution electron cryomicroscopy and X-ray crystallography are revealing the structure of this important molecular machine. Both approaches have contributed to our understanding of the organization of the transmembrane helices of higher plant and cyanobacterial PS II and both indicate that PS II normally functions as a dimer. However the high-resolution electron density maps derived from X-ray crystallography currently at 3·7/3·8 Å, have allowed assignments to be made to the redox active cofactors involved in the light-driven water–plastoquinone oxidoreductase activity and to the chlorophyll molecules that absorb and transfer energy to the reaction centre. In particular the X-ray work has identified density that can accommodate the four manganese atoms which catalyse the water-oxidation process. The Mn cluster is located at the lumenal surface of the D1 protein and approximately 7 Å from the redox active tyrosine residue (YZ) which acts an electron/proton transfer link to the primary oxidant P680.+. The lower resolution electron microscopy studies, however, are providing structural models of larger PS II supercomplexes that are ideal frameworks in which to incorporate the X-ray derived structures.

scholarly journals On the Consistency of QCBED Structure Factor Measurements for TiO2 (Rutile)

2003 ◽  
Vol 9 (5) ◽  
pp. 457-467 ◽  
Author(s):  
Bin Jiang ◽  
Jian-Min Zuo ◽  
Jesper Friis ◽  
John C.H. Spence
Keyword(s):  
Structure Factor ◽  
Bragg Reflection ◽  
Surface Oxidation ◽  
Ccd Camera ◽  
Order Structure ◽  
X Ray ◽  
X Ray Crystallography ◽  
Order Of Magnitude ◽  
Factor Measurement ◽  
Bond Charge

The same Bragg reflection in TiO2 from 12 different (CBED) patterns (from different crystals, orientations, and thicknesses) are analyzed quantitatively to evaluate the consistency of the quantitative CBED method for bond-charge mapping. The standard deviation in the resulting distribution of derived X-ray structure factors is found to be an order of magnitude smaller than that in conventional X-ray work, and the standard error (0.026% for FX(110)) is slightly better than obtained by the X-ray Pendellösung method applied to silicon. This is sufficiently accurate to distinguish between atomic, covalent, and ionic models of bonding. We describe the importance of extracting experimental parameters from CCD camera characterization, and of surface oxidation and crystal shape. The current experiments show that the QCBED method is now a robust and powerful tool for low-order structure factor measurement, which does not suffer from the large extinction (multiple scattering) errors that occur in inorganic X-ray crystallography, and may be applied to nanocrystals. Our results will be used to understand the role of d-electrons in the chemical bonding of TiO2.

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