scholarly journals Glutathione binding to the plant At Atm3 transporter and implications for the conformational coupling of ABC transporters

2021 ◽  
Author(s):  
Chengcheng Fan ◽  
Douglas C Rees
Keyword(s):  
Abc Transporters ◽  
Ternary Complex ◽  
Conformational States ◽  
Type Iv ◽  
Structural Framework ◽  
Cryo Electron Microscopy ◽  
Heavy Metal Detoxification ◽  
Transport Cycle ◽  
Mixed Disulfides ◽  
Glutathione Binding

The ATP Binding Cassette (ABC) transporter of mitochondria (Atm) from Arabidopsis thaliana (AtAtm3) has been implicated in the maturation of cytosolic iron-sulfur proteins and heavy metal detoxification, plausibly by exporting glutathione derivatives. Using single-particle cryo-electron microscopy, we have determined structures of AtAtm3 in multiple conformational states. These structures not only provide a structural framework for defining the alternating access transport cycle, but also highlight an unappreciated feature of the glutathione binding site, namely the paucity of cysteine residues that could potentially form inhibitory mixed disulfides with glutathione. Despite extensive efforts, we were unable to prepare the ternary complex of AtAtm3 with bound GSSG and MgATP. A survey of structurally characterized type IV ABC transporters that includes AtAtm3 establishes that while nucleotides are found associated with all conformational states, they are effectively required to stabilize occluded and outward-facing conformations. In contrast, transport substrates have only been observed associated with inward-facing conformations. The absence of structures containing both nucleotide and transport substrate suggests that this ternary complex exists only transiently during the transport cycle.

Watching conformational dynamics of ABC transporters with single-molecule tools

2015 ◽  
Vol 43 (5) ◽  
pp. 1041-1047 ◽  
Author(s):  
Florence Husada ◽  
Giorgos Gouridis ◽  
Ruslan Vietrov ◽  
Gea K. Schuurman-Wolters ◽  
Evelyn Ploetz ◽  
...  
Keyword(s):  
Single Molecule ◽  
Abc Transporters ◽  
Fluorescent Dyes ◽  
Substrate Binding ◽  
Conformational Dynamics ◽  
Cell Volume Regulation ◽  
Size Exclusion ◽  
Conformational States ◽  
Functional Interpretation ◽  
Transport Cycle

ATP-binding cassette (ABC) transporters play crucial roles in cellular processes, such as nutrient uptake, drug resistance, cell-volume regulation and others. Despite their importance, all proposed molecular models for transport are based on indirect evidence, i.e. functional interpretation of static crystal structures and ensemble measurements of function and structure. Thus, classical biophysical and biochemical techniques do not readily visualize dynamic structural changes. We recently started to use single-molecule fluorescence techniques to study conformational states and changes of ABC transporters in vitro, in order to observe directly how the different steps during transport are coordinated. This review summarizes our scientific strategy and some of the key experimental advances that allowed the substrate-binding mechanism of prokaryotic ABC importers and the transport cycle to be explored. The conformational states and transitions of ABC-associated substrate-binding domains (SBDs) were visualized with single-molecule FRET, permitting a direct correlation of structural and kinetic information of SBDs. We also delineated the different steps of the transport cycle. Since information in such assays are restricted by proper labelling of proteins with fluorescent dyes, we present a simple approach to increase the amount of protein with FRET information based on non-specific interactions between a dye and the size-exclusion chromatography (SEC) column material used for final purification.

scholarly journals A structural framework for unidirectional transport by a bacterial ABC exporter

2020 ◽  
Vol 117 (32) ◽  
pp. 19228-19236
Author(s):  
Chengcheng Fan ◽  
Jens T. Kaiser ◽  
Douglas C. Rees
Keyword(s):  
Conformational Changes ◽  
Iron Homeostasis ◽  
Atp Hydrolysis ◽  
Transmembrane Helix ◽  
Reverse Direction ◽  
Conformational States ◽  
X Ray Crystallography ◽  
Binding Domains ◽  
Structural Framework ◽  
Glutathione Derivatives

The ATP-binding cassette (ABC) transporter of mitochondria (Atm1) mediates iron homeostasis in eukaryotes, while the prokaryotic homolog fromNovosphingobium aromaticivorans(NaAtm1) can export glutathione derivatives and confer protection against heavy-metal toxicity. To establish the structural framework underlying theNaAtm1 transport mechanism, we determined eight structures by X-ray crystallography and single-particle cryo-electron microscopy in distinct conformational states, stabilized by individual disulfide crosslinks and nucleotides. AsNaAtm1 progresses through the transport cycle, conformational changes in transmembrane helix 6 (TM6) alter the glutathione-binding site and the associated substrate-binding cavity. Significantly, kinking of TM6 in the post-ATP hydrolysis state stabilized by MgADPVO4eliminates this cavity, precluding uptake of glutathione derivatives. The presence of this cavity during the transition from the inward-facing to outward-facing conformational states, and its absence in the reverse direction, thereby provide an elegant and conceptually simple mechanism for enforcing the export directionality of transport byNaAtm1. One of the disulfide crosslinkedNaAtm1 variants characterized in this work retains significant glutathione transport activity, suggesting that ATP hydrolysis and substrate transport by Atm1 may involve a limited set of conformational states with minimal separation of the nucleotide-binding domains in the inward-facing conformation.

scholarly journals Cryo-electron Microscopy Structure and Transport Mechanism of a Wall Teichoic Acid ABC Transporter

mBio ◽  
2020 ◽  
Vol 11 (2) ◽  
Author(s):  
Li Chen ◽  
Wen-Tao Hou ◽  
Tao Fan ◽  
Banghui Liu ◽  
Ting Pan ◽  
...  
Keyword(s):  
Abc Transporter ◽  
Conformational Changes ◽  
Abc Transporters ◽  
Rational Design ◽  
Teichoic Acid ◽  
Inhibitory Mechanism ◽  
Wall Teichoic Acid ◽  
Content Type ◽  
Design And Optimization ◽  
Cryo Electron Microscopy

ABSTRACT The wall teichoic acid (WTA) is a major cell wall component of Gram-positive bacteria, such as methicillin-resistant Staphylococcus aureus (MRSA), a common cause of fatal clinical infections in humans. Thus, the indispensable ABC transporter TarGH, which flips WTA from cytoplasm to extracellular space, becomes a promising target of anti-MRSA drugs. Here, we report the 3.9-Å cryo-electron microscopy (cryo-EM) structure of a 50% sequence-identical homolog of TarGH from Alicyclobacillus herbarius at an ATP-free and inward-facing conformation. Structural analysis combined with activity assays enables us to clearly decode the binding site and inhibitory mechanism of the anti-MRSA inhibitor Targocil, which targets TarGH. Moreover, we propose a “crankshaft conrod” mechanism utilized by TarGH, which can be applied to similar ABC transporters that translocate a rather big substrate through relatively subtle conformational changes. These findings provide a structural basis for the rational design and optimization of antibiotics against MRSA. IMPORTANCE The wall teichoic acid (WTA) is a major component of cell wall and a pathogenic factor in methicillin-resistant Staphylococcus aureus (MRSA). The ABC transporter TarGH is indispensable for flipping WTA precursor from cytoplasm to the extracellular space, thus making it a promising drug target for anti-MRSA agents. The 3.9-Å cryo-EM structure of a TarGH homolog helps us to decode the binding site and inhibitory mechanism of a recently reported inhibitor, Targocil, and provides a structural platform for rational design and optimization of potential antibiotics. Moreover, we propose a “crankshaft conrod” mechanism to explain how a big substrate is translocated through subtle conformational changes of type II exporters. These findings advance our understanding of anti-MRSA drug design and ABC transporters.

scholarly journals Pathogenic siderophore ABC importer YbtPQ adopts a surprising fold of exporter

2020 ◽  
Vol 6 (6) ◽  
pp. eaay7997 ◽  
Author(s):  
Zhiming Wang ◽  
Wenxin Hu ◽  
Hongjin Zheng
Keyword(s):  
Bound States ◽  
Molecular Mechanisms ◽  
Transmembrane Helix ◽  
Human Pathogens ◽  
Binding Domains ◽  
Type Iv ◽  
Cryo Electron Microscopy ◽  
Open Conformation ◽  
Multiple Structures ◽  
First Time

To fight for essential metal ions, human pathogens secrete virulence-associated siderophores and retake the metal-chelated siderophores through a subfamily of adenosine triphosphate (ATP)–binding cassette (ABC) importer, whose molecular mechanisms are completely unknown. We have determined multiple structures of the yersiniabactin importer YbtPQ from uropathogenic Escherichia coli (UPEC) at inward-open conformation in both apo and substrate-bound states by cryo–electron microscopy. YbtPQ does not adopt any known fold of ABC importers but surprisingly adopts the fold of type IV ABC exporters. To our knowledge, it is the first time an exporter fold of ABC importer has been reported. We have also observed two unique features in YbtPQ: unwinding of a transmembrane helix in YbtP upon substrate release and tightly associated nucleotide-binding domains without bound nucleotides. Together, our study suggests that siderophore ABC importers have a distinct transport mechanism and should be classified as a separate subfamily of ABC importers.

scholarly journals Mechanistic origin of the sigmoidal rate behaviour of glucokinase

1986 ◽  
Vol 233 (2) ◽  
pp. 347-350 ◽  
Author(s):  
G Pettersson
Keyword(s):  
Ternary Complex ◽  
Flow Characteristics ◽  
Free Enzyme ◽  
Kinetic Properties ◽  
Complex Mechanism ◽  
Insufficient Evidence ◽  
Conformational States ◽  
Model Studies ◽  
Glucose Binding ◽  
Rate Behaviour

Model studies are presented which demonstrate that reactions proceeding by a random ternary-complex mechanism may exhibit most pronounced deviations from Michaelis-Menten kinetics even if the reaction is effectively ordered with respect to net reaction flow. In particular, the kinetic properties and reaction flow characteristics of glucokinase can be accounted for in such terms. It is concluded that insufficient evidence has been presented to support the idea that glucokinase operates by a ‘mnemonical’ type of mechanism involving glucose binding to distinct conformational states of free enzyme. The sigmoidal rate behaviour of glucokinase can presently be more simply explained in terms of glucose binding to differently ligated states of the enzyme.

Functions and interplay of the tRNA-binding sites of the ribosome

2002 ◽  
Vol 30 (2) ◽  
pp. 133-140 ◽  
Author(s):  
V. Márquez ◽  
D. N. Wilson ◽  
K. H. Nierhaus
Keyword(s):  
Binding Sites ◽  
Genetic Information ◽  
Small Angle Neutron Scattering ◽  
Ternary Complex ◽  
Large Body ◽  
Reading Frame ◽  
X Ray ◽  
Cryo Electron Microscopy ◽  
Precise Movement ◽  
Trna Binding

The ribosome translates the genetic information of an mRNA molecule into a sequence of amino acids. The ribosome utilizes tRNAs to connect elements of the RNA and protein worlds during protein synthesis, i.e. an anticodon as a unit of genetic information with the corresponding amino acid as a building unit of proteins. Three tRNA-binding sites are located on the ribosome, termed the A, P and E sites. In recent years the tRNA-binding sites have been localized on the ribosome by three different techniques, small-angle neutron scattering, cryo-electron microscopy and X-ray analyses of 70 S crystals. These high-resolution glimpses into various ribosomal states together with a large body of biochemical data reveal an intricate interplay between the tRNAs and the three ribosomal binding sites, providing an explanation for the remarkable features of the ribosome, such as the ability to select the correct ternary complex aminoacyl-tRNA · EF-Tu · GTP out of more than 40 extremely similar tRNA complexes, the precise movement of the tRNA2 · mRNA complex during translocation and the maintenance of the reading frame.

scholarly journals Structural basis of trehalose recycling by the ABC transporter LpqY-SugABC

2020 ◽  
Vol 6 (44) ◽  
pp. eabb9833
Author(s):  
Fengjiang Liu ◽  
Jingxi Liang ◽  
Bing Zhang ◽  
Yan Gao ◽  
Xiuna Yang ◽  
...  
Keyword(s):  
Electron Microscopy ◽  
Abc Transporter ◽  
Abc Transporters ◽  
Unique Feature ◽  
Structural Basis ◽  
Human Pathogens ◽  
Gram Positive Bacteria ◽  
Cryo Electron Microscopy ◽  
Uptake Of Nutrients ◽  
Initial Mode

In bacteria, adenosine 5′-triphosphate (ATP)–binding cassette (ABC) importers are essential for the uptake of nutrients including the nonreducing disaccharide trehalose, a metabolite that is crucial for the survival and virulence of several human pathogens including Mycobacterium tuberculosis. SugABC is an ABC transporter that translocates trehalose from the periplasmic lipoprotein LpqY into the cytoplasm of mycobacteria. Here, we report four high-resolution cryo–electron microscopy structures of the mycobacterial LpqY-SugABC complex to reveal how it binds and passes trehalose through the membrane to the cytoplasm. A unique feature observed in this system is the initial mode of capture of the trehalose at the LpqY interface. Uptake is achieved by a pivotal rotation of LpqY relative to SugABC, moving from an open and accessible conformation to a clamped conformation upon trehalose binding. These findings enrich our understanding as to how ABC transporters facilitate substrate transport across the membrane in Gram-positive bacteria.

scholarly journals Type IV Pilus Structure by Cryo-Electron Microscopy and Crystallography: Implications for Pilus Assembly and Functions

2006 ◽  
Vol 23 (5) ◽  
pp. 651-662 ◽  
Author(s):  
Lisa Craig ◽  
Niels Volkmann ◽  
Andrew S. Arvai ◽  
Michael E. Pique ◽  
Mark Yeager ◽  
...  
Keyword(s):  
Electron Microscopy ◽  
Type Iv Pilus ◽  
Type Iv ◽  
Cryo Electron Microscopy ◽  
Pilus Assembly
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