scholarly journals Chemo-mechanical Coupling in the Transport Cycle of a Type II ABC Transporter

2018 ◽  
Author(s):  
Koichi Tamura ◽  
Hiroshi Sugimoto ◽  
Yoshitsugu Shiro ◽  
Yuji Sugita
Keyword(s):  
Atpase Activity ◽  
Abc Transporter ◽  
Conformational Changes ◽  
Abc Transporters ◽  
Md Simulations ◽  
Coupling Mechanism ◽  
Transmembrane Helices ◽  
Mechanical Coupling ◽  
Binding Domains ◽  
Wide Range

AbstractAT P -binding cassette (ABC) transporters are integral membrane proteins that translocate a wide range of substrates across biological membranes, harnessing free energy from the binding and hydrolysis of ATP. To understand the mechanism of the inward- to outward-facing transition that could be achieved by tight regulation of ATPase activity through extensive conformational changes of the protein, we applied template-based iterative all-atom molecular dynamics (MD) simulation to the heme ABC transporter BhuUV-T. The simulations, together with biased MDs, predict two new conformations of the protein, namely, occluded (Occ) and outward-facing (OF) conformations. The comparison between the inward-facing crystal structure and the predicted two structures shows atomic details of the gating motions at the transmembrane helices and dimerization of the nucleotide-binding domains (NBDs). The MD simulations further reveal a novel role of the ABC signature motifs (LSGG[Q/E]) at the NBDs in decelerating ATPase activity in the Occ form through sporadic flipping of the side chains of the LSGG[Q/E] catalytic serine residues. The orientational changes are coupled to loose NBD dimerization in the Occ state, whereas they are blocked in the OF form where the NBDs are tightly dimerized. The chemo-mechanical coupling mechanism may apply to other types of ABC transporters having the conserved LSGG[Q/E] signature motifs.

scholarly journals Substrate recognition and ATPase activity of the E. coli cysteine/cystine ABC transporter YecSC-FliY

2020 ◽  
Vol 295 (16) ◽  
pp. 5245-5256 ◽  
Author(s):  
Siwar Sabrialabed ◽  
Janet G. Yang ◽  
Elon Yariv ◽  
Nir Ben-Tal ◽  
Oded Lewinson
Keyword(s):  
Atpase Activity ◽  
Abc Transporter ◽  
Conformational Changes ◽  
Binding Protein ◽  
Substrate Binding ◽  
Transporter Family ◽  
Wide Range ◽  
Substrate Binding Protein ◽  
Sulfur Containing ◽  
Sulfur Containing Compounds

Sulfur is essential for biological processes such as amino acid biogenesis, iron–sulfur cluster formation, and redox homeostasis. To acquire sulfur-containing compounds from the environment, bacteria have evolved high-affinity uptake systems, predominant among which is the ABC transporter family. Theses membrane-embedded enzymes use the energy of ATP hydrolysis for transmembrane transport of a wide range of biomolecules against concentration gradients. Three distinct bacterial ABC import systems of sulfur-containing compounds have been identified, but the molecular details of their transport mechanism remain poorly characterized. Here we provide results from a biochemical analysis of the purified Escherichia coli YecSC-FliY cysteine/cystine import system. We found that the substrate-binding protein FliY binds l-cystine, l-cysteine, and d-cysteine with micromolar affinities. However, binding of the l- and d-enantiomers induced different conformational changes of FliY, where the l- enantiomer–substrate-binding protein complex interacted more efficiently with the YecSC transporter. YecSC had low basal ATPase activity that was moderately stimulated by apo FliY, more strongly by d-cysteine–bound FliY, and maximally by l-cysteine– or l-cystine–bound FliY. However, at high FliY concentrations, YecSC reached maximal ATPase rates independent of the presence or nature of the substrate. These results suggest that FliY exists in a conformational equilibrium between an open, unliganded form that does not bind to the YecSC transporter and closed, unliganded and closed, liganded forms that bind this transporter with variable affinities but equally stimulate its ATPase activity. These findings differ from previous observations for similar ABC transporters, highlighting the extent of mechanistic diversity in this large protein family.

scholarly journals Insect ATP-Binding Cassette (ABC) Transporters: Roles in Xenobiotic Detoxification and Bt Insecticidal Activity

2019 ◽  
Vol 20 (11) ◽  
pp. 2829 ◽  
Author(s):  
Chao Wu ◽  
Swapan Chakrabarty ◽  
Minghui Jin ◽  
Kaiyu Liu ◽  
Yutao Xiao
Keyword(s):  
Abc Transporter ◽  
Conformational Changes ◽  
Abc Transporters ◽  
Insecticidal Activity ◽  
Atp Hydrolysis ◽  
Atp Binding ◽  
Bt Toxin ◽  
Xenobiotic Detoxification ◽  
Binding Domains ◽  
Atp Binding Cassette

ATP-binding cassette (ABC) transporters, a large class of transmembrane proteins, are widely found in organisms and play an important role in the transport of xenobiotics. Insect ABC transporters are involved in insecticide detoxification and Bacillus thuringiensis (Bt) toxin perforation. The complete ABC transporter is composed of two hydrophobic transmembrane domains (TMDs) and two nucleotide binding domains (NBDs). Conformational changes that are needed for their action are mediated by ATP hydrolysis. According to the similarity among their sequences and organization of conserved ATP-binding cassette domains, insect ABC transporters have been divided into eight subfamilies (ABCA–ABCH). This review describes the functions and mechanisms of ABC transporters in insecticide detoxification, plant toxic secondary metabolites transport and insecticidal activity of Bt toxin. With improved understanding of the role and mechanisms of ABC transporter in resistance to insecticides and Bt toxins, we can identify valuable target sites for developing new strategies to control pests and manage resistance and achieve green pest control.

A long road towards the structure of respiratory complex I, a giant molecular proton pump

2013 ◽  
Vol 41 (5) ◽  
pp. 1265-1271 ◽  
Author(s):  
Leonid A. Sazanov ◽  
Rozbeh Baradaran ◽  
Rouslan G. Efremov ◽  
John M. Berrisford ◽  
Gurdeep Minhas
Keyword(s):  
Conformational Changes ◽  
Complex I ◽  
Thermus Thermophilus ◽  
Proton Translocation ◽  
Nadh:Ubiquinone Oxidoreductase ◽  
Coupling Mechanism ◽  
Membrane Domains ◽  
Transmembrane Helices ◽  
Inner Mitochondrial Membrane ◽  
Wide Range

Complex I (NADH:ubiquinone oxidoreductase) is central to cellular energy production, being the first and largest enzyme of the respiratory chain in mitochondria. It couples electron transfer from NADH to ubiquinone with proton translocation across the inner mitochondrial membrane and is involved in a wide range of human neurodegenerative disorders. Mammalian complex I is composed of 44 different subunits, whereas the ‘minimal’ bacterial version contains 14 highly conserved ‘core’ subunits. The L-shaped assembly consists of hydrophilic and membrane domains. We have determined all known atomic structures of complex I, starting from the hydrophilic domain of Thermus thermophilus enzyme (eight subunits, nine Fe–S clusters), followed by the membrane domains of the Escherichia coli (six subunits, 55 transmembrane helices) and T. thermophilus (seven subunits, 64 transmembrane helices) enzymes, and finally culminating in a recent crystal structure of the entire intact complex I from T. thermophilus (536 kDa, 16 subunits, nine Fe–S clusters, 64 transmembrane helices). The structure suggests an unusual and unique coupling mechanism via long-range conformational changes. Determination of the structure of the entire complex was possible only through this step-by-step approach, building on from smaller subcomplexes towards the entire assembly. Large membrane proteins are notoriously difficult to crystallize, and so various non-standard and sometimes counterintuitive approaches were employed in order to achieve crystal diffraction to high resolution and solve the structures. These steps, as well as the implications from the final structure, are discussed in the present review.

scholarly journals Combining Mutations That Inhibit Two Distinct Steps of the ATP Hydrolysis Cycle Restores Wild-Type Function in the Lipopolysaccharide Transporter and Shows that ATP Binding Triggers Transport

mBio ◽  
2019 ◽  
Vol 10 (4) ◽  
Author(s):  
Brent W. Simpson ◽  
Karanbir S. Pahil ◽  
Tristan W. Owens ◽  
Emily A. Lundstedt ◽  
Rebecca M. Davis ◽  
...  
Keyword(s):  
Abc Transporter ◽  
Conformational Changes ◽  
Abc Transporters ◽  
Atp Binding ◽  
Gram Negative Bacteria ◽  
Wild Type ◽  
Lethal Effects ◽  
Gram Negative ◽  
Binding Domains ◽  
Transport Cycle

ABSTRACT ATP-binding cassette (ABC) transporters constitute a large family of proteins present in all domains of life. They are powered by dynamic ATPases that harness energy from binding and hydrolyzing ATP through a cycle that involves the closing and reopening of their two ATP-binding domains. The LptB2FGC exporter is an essential ABC transporter that assembles lipopolysaccharides (LPS) on the surface of Gram-negative bacteria to form a permeability barrier against many antibiotics. LptB2FGC extracts newly synthesized LPS molecules from the inner membrane and powers their transport across the periplasm and through the outer membrane. How LptB2FGC functions remains poorly understood. Here, we show that the C-terminal domain of the dimeric LptB ATPase is essential for LPS transport in Escherichia coli. Specific changes in the C-terminal domain of LptB cause LPS transport defects that can be repaired by intragenic suppressors altering the ATP-binding domains. Surprisingly, we found that each of two lethal changes in the ATP-binding and C-terminal domains of LptB, when present in combined form, suppressed the defects associated with the other to restore LPS transport to wild-type levels both in vivo and in vitro. We present biochemical evidence explaining the effect that each of these mutations has on LptB function and how the observed cosuppression results from the opposing lethal effects these changes have on the dimerization state of the LptB ATPase. We therefore propose that these sites modulate the closing and reopening of the LptB dimer, providing insight into how the LptB2FGC transporter cycles to export LPS to the cell surface and how to inhibit this essential envelope biogenesis process. IMPORTANCE Gram-negative bacteria are naturally resistant to many antibiotics because their surface is covered by the glycolipid LPS. Newly synthesized LPS is transported across the cell envelope by the multiprotein Lpt machinery, which includes LptB2FGC, an unusual ABC transporter that extracts LPS from the inner membrane. Like in other ABC transporters, the LptB2FGC transport cycle is driven by the cyclical conformational changes that a cytoplasmic, dimeric ATPase, LptB, undergoes when binding and hydrolyzing ATP. How these conformational changes are controlled in ABC transporters is poorly understood. Here, we identified two lethal changes in LptB that, when combined, remarkably restore wild-type transport function. Biochemical studies revealed that the two changes affect different steps in the transport cycle, having opposing, lethal effects on LptB’s dimerization cycle. Our work provides mechanistic details about the LptB2FGC extractor that could be used to develop Lpt inhibitors that would overcome the innate antibiotic resistance of Gram-negative bacteria.

Investigating the dynamic nature of the ABC transporters: ABCB1 and MsbA as examples for the potential synergies of MD theory and EPR applications

2015 ◽  
Vol 43 (5) ◽  
pp. 1023-1032 ◽  
Author(s):  
Thomas Stockner ◽  
Anna Mullen ◽  
Fraser MacMillan
Keyword(s):  
Crystal Structures ◽  
Conformational Changes ◽  
Abc Transporters ◽  
Epr Spectroscopy ◽  
Structural Information ◽  
Dynamic Properties ◽  
Molecular System ◽  
Synergistic Effects ◽  
Md Simulations ◽  
Substrate Spectrum

ABC transporters are primary active transporters found in all kingdoms of life. Human multidrug resistance transporter ABCB1, or P-glycoprotein, has an extremely broad substrate spectrum and confers resistance against chemotherapy drug treatment in cancer cells. The bacterial ABC transporter MsbA is a lipid A flippase and a homolog to the human ABCB1 transporter, with which it partially shares its substrate spectrum. Crystal structures of MsbA and ABCB1 have been solved in multiple conformations, providing a glimpse into the possible conformational changes the transporter could be going through during the transport cycle. Crystal structures are inherently static, while a dynamic picture of the transporter in motion is needed for a complete understanding of transporter function. Molecular dynamics (MD) simulations and electron paramagnetic resonance (EPR) spectroscopy can provide structural information on ABC transporters, but the strength of these two methods lies in the potential to characterise the dynamic regime of these transporters. Information from the two methods is quite complementary. MD simulations provide an all atom dynamic picture of the time evolution of the molecular system, though with a narrow time window. EPR spectroscopy can probe structural, environmental and dynamic properties of the transporter in several time regimes, but only through the attachment sites of an exogenous spin label. In this review the synergistic effects that can be achieved by combining the two methods are highlighted, and a brief methodological background is also presented.

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.

ATP-Induced Conformational Changes of Nucleotide-Binding Domains in an ABC Transporter. Importance of the Water-Mediated Entropic Force

2014 ◽  
Vol 118 (44) ◽  
pp. 12612-12620 ◽  
Author(s):  
Tomohiko Hayashi ◽  
Shuntaro Chiba ◽  
Yusuke Kaneta ◽  
Tadaomi Furuta ◽  
Minoru Sakurai
Keyword(s):  
Abc Transporter ◽  
Conformational Changes ◽  
Nucleotide Binding ◽  
Entropic Force ◽  
Binding Domains

scholarly journals Role of the ABC transporter TruMDR2 in terbinafine, 4-nitroquinoline N-oxide and ethidium bromide susceptibility in Trichophyton rubrum

2006 ◽  
Vol 55 (8) ◽  
pp. 1093-1099 ◽  
Author(s):  
Ana Lúcia Fachin ◽  
Monica S. Ferreira-Nozawa ◽  
Walter Maccheroni ◽  
Nilce M. Martinez-Rossi
Keyword(s):  
Abc Transporter ◽  
Ethidium Bromide ◽  
Abc Transporters ◽  
Trichophyton Rubrum ◽  
Single Copy ◽  
Drug Susceptibility ◽  
Transmembrane Segments ◽  
Binding Domains ◽  
Abc Protein ◽  
Identical Nucleotide

A single-copy gene, designated TruMDR2, encoding an ATP-binding cassette (ABC) transporter was cloned and sequenced from the dermatophyte Trichophyton rubrum. The ORF of TruMDR2 was 4048 nt and the deduced amino acid sequence showed high homology with ABC transporters involved in drug efflux in other fungi. The encoded ABC protein predicted 12 transmembrane segments (TMSs) and two almost identical nucleotide-binding domains (NBDs) arranged in two halves in a (TMS6–NBD)2 configuration and could be classified as a member of the multidrug-resistance (MDR) class of ABC transporters. Northern blot analyses revealed an increased level of transcription of the TruMDR2 gene when mycelium was exposed to acriflavine, benomyl, ethidium bromide, ketoconazole, chloramphenicol, griseofulvin, fluconazole, imazalil, itraconazole, methotrexate, 4-nitroquinoline N-oxide (4NQO) or tioconazole. Disruption of the TruMDR2 gene rendered the mutant more sensitive to terbinafine, 4NQO and ethidium bromide than the control strain, suggesting that this transporter plays a role in modulating drug susceptibility in T. rubrum.

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