scholarly journals The uncoupled ATPase activity of the ABC transporter BtuC2D2 leads to a hysteretic conformational change, conformational memory and improved activity

2016 ◽  
Vol 6 (1) ◽  
Author(s):  
Nurit Livnat-Levanon ◽  
Amy I. Gilson ◽  
Nir Ben-Tal ◽  
Oded Lewinson
Keyword(s):  
Atpase Activity ◽  
Conformational Change ◽  
Abc Transporter ◽  
Conformational Memory

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 Specific Molecular Chaperone Interactions and an ATP-dependent Conformational Change Are Required during Posttranslational Protein Translocation into the Yeast ER

1998 ◽  
Vol 9 (12) ◽  
pp. 3533-3545 ◽  
Author(s):  
Amie J. McClellan ◽  
James B. Endres ◽  
Joseph P. Vogel ◽  
Debra Palazzi ◽  
Mark D. Rose ◽  
...  
Keyword(s):  
Atpase Activity ◽  
Conformational Change ◽  
Molecular Chaperone ◽  
Atp Hydrolysis ◽  
Protein Translocation ◽  
Wild Type ◽  
J Domain ◽  
Chaperone Interactions ◽  
Dependent Interaction ◽  
Er Membrane

The posttranslational translocation of proteins across the endoplasmic reticulum (ER) membrane in yeast requires ATP hydrolysis and the action of hsc70s (DnaK homologues) and DnaJ homologues in both the cytosol and ER lumen. Although the cytosolic hsc70 (Ssa1p) and the ER lumenal hsc70 (BiP) are homologous, they cannot substitute for one another, possibly because they interact with specific DnaJ homologues on each side of the ER membrane. To investigate this possibility, we purified Ssa1p, BiP, Ydj1p (a cytosolic DnaJ homologue), and a GST–63Jp fusion protein containing the lumenal DnaJ region of Sec63p. We observed that BiP, but not Ssa1p, is able to associate with GST–63Jp and that Ydj1p stimulates the ATPase activity of Ssa1p up to 10-fold but increases the ATPase activity of BiP by <2-fold. In addition, Ydj1p and ATP trigger the release of an unfolded polypeptide from Ssa1p but not from BiP. To understand further how BiP drives protein translocation, we purified four dominant lethal mutants of BiP. We discovered that each mutant is defective for ATP hydrolysis, fails to undergo an ATP-dependent conformational change, and cannot interact with GST–63Jp. Measurements of protein translocation into reconstituted proteoliposomes indicate that the mutants inhibit translocation even in the presence of wild-type BiP. We conclude that a conformation- and ATP-dependent interaction of BiP with the J domain of Sec63p is essential for protein translocation and that the specificity of hsc70 action is dictated by their DnaJ partners.

ATPase activity and ATP/ADP-induced conformational change in the bacterial toxin exporter hemolysin B

1993 ◽  
Vol 21 (4) ◽  
pp. 347S-347S ◽  
Author(s):  
IRINA MILISAV-RIBARIC ◽  
COLIN HUGHES ◽  
EVA KORONAKIS ◽  
VASSILIS KORONAKIS
Keyword(s):  
Atpase Activity ◽  
Conformational Change ◽  
Bacterial Toxin

Effects of physical training on cardiac myosin ATPase activity

1975 ◽  
Vol 228 (4) ◽  
pp. 1178-1182 ◽  
Author(s):  
AK Bhan ◽  
J Scheuer
Keyword(s):  
Ethylene Glycol ◽  
Atpase Activity ◽  
Conformational Change ◽  
Physical Training ◽  
Active Site ◽  
Myosin Atpase ◽  
Control Group ◽  
Cardiac Myosin ◽  
Heavy Meromyosin ◽  
Myosin Atpase Activity

Cardiac myosin from rats exercised 90 or 150 min daily for 8 wk was compared with the myosin from the hearts of matched sedentary controls. The Ca++-ATPase activity was increased 17 percent in rats exercised 90 min and 30 percent in rats exercised 150 min daily. In the exercised group 0.18 M KCl increased the myosin ATPase activity by 50 percent but had no effect in the control group. Ethylene glycol activated the Ca++-ATPase in control myosin preparations, but had no significant effect on myosin from conditioned hearts. Heavy meromyosin (HMM) from conditioned hearts had a higher Ca++-ATPase activity than from controls. Fluorescence with 8-anilinonaphthalene sulfonate (ANS) was increased 30 percent in HMM from conditioned hearts. The results suggest that the increased myosin ATPase activity in the hearts of exercised animals may be due to a local conformational change at or near the active site.

scholarly journals LptB2FG is an ABC transporter with Adenylate Kinase activity regulated by LptC/A recruitment

2021 ◽  
Author(s):  
Tiago Baeta ◽  
Karine Giandoreggio-Barranco ◽  
Isabel Ayala ◽  
Elisabete CCM Moura ◽  
Paola Sperandeo ◽  
...  
Keyword(s):  
Atpase Activity ◽  
Abc Transporter ◽  
Adenylate Kinase ◽  
Kinase Activity ◽  
Cell Envelope ◽  
Transmembrane Helix ◽  
Gram Negative Bacteria ◽  
Gram Negative ◽  
Entire Cell ◽  
Lipid Environment

Lipopolysaccharide (LPS) is an essential glycolipid covering the surface of gram-negative bacteria. Its transport involves a dedicated 7 protein transporter system, the Lpt machinery, that physically spans the entire cell envelope. LptB2FG complex is an ABC transporter that hydrolyses Adenosine Triphosphate (ATP) to extract LPS from the inner membrane (IM). LptB2FG was extracted directly from IM with its original lipid environment by Styrene-Maleic acids polymers(SMA). SMA-LptB2FG in nanodiscs displays ATPase activity and a previously uncharacterized Adenylate Kinase (AK) activity. It catalyzes phosphotransfer between two ADP molecules to generate ATP and AMP. ATPase and AK activities of LptB2FG are both stimulated by the interaction on the periplasmic side with LptC and LptA partners and inhibited by the presence of LptC transmembrane helix. Isolated ATPase module (LptB) has weak AK activity in absence of LptF and LptG, and one mutation, that weakens affinity for ADP, has AK activity similar to that of fully assembled complex. LptB2FG is thus capable of producing ATP from ADP depending on the assembly of the Lpt bridge and the AK activity might be important to ensure efficient LPS transport in fully assembled Lpt system.

PK11195, a Peripheral Benzodiazepine Receptor (PBR) Ligand, Broadly Blocks Drug Efflux to Chemosensitize Leukemia and Myeloma Cells by a PBR-Independent, Direct Transporter-Modulating Mechanism.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 1533-1533
Author(s):  
Roland B. Walter ◽  
Jason Pirga ◽  
Michelle R. Cronk ◽  
Sasha J. Mayer ◽  
Frederick R. Appelbaum ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
Multidrug Resistance ◽  
Atpase Activity ◽  
Cell Lines ◽  
Abc Transporter ◽  
Ectopic Expression ◽  
Benzodiazepine Receptor ◽  
Drug Efflux ◽  
Peripheral Benzodiazepine Receptor ◽  
Resistance Protein

Abstract Background: Multidrug resistance (MDR) is frequently associated with expression of anti-apoptotic proteins of the Bcl-2 family and/or ATP-binding cassette (ABC) transporter proteins. We previously showed that the peripheral benzodiazepine receptor (pBR) ligand, PK11195, promotes mitochondrial apoptosis and blocks P-glycoprotein (Pgp)-mediated drug efflux, establishing PK11195 as a promising MDR reversal agent. We have now assessed its effect on other ABC transporters, namely multidrug resistance protein (MRP) and breast cancer resistance protein (BCRP), and have explored the mechanism by which PK11195 blocks drug efflux, focusing on Pgp as a paradigm. Methods: Flow cytometry was used to measure efflux of the cytotoxic drug, mitoxantrone (MIT), that was inhibited by cyclosporine A (CSA; to assess Pgp function), MK-571 (to assess MRP function), or Ko143 or GF120918 (to assess BCRP function). MIT-induced toxicity was determined after 24 hours by flow cytometry with DiOC6(3) plus propidium iodide staining, or by 3H-thymidine incorporation. Hematopoietic cell lines were virally transduced with human pBR to assess the effect of pBR expression on PK11195 action. Specific 3H-PK11195 binding was determined by competition with 1000-fold excess unlabeled PK11195. Microsome and plasma membranes were prepared using sucrose gradients. ATPase activity was measured as the liberation of inorganic phosphate detected by absorbance at 800nM. Conformation-specific and conformation-insensitive anti-Pgp monoclonal antibodies were used to analyze PK11195 effects on Pgp by flow cytometry. Results: Using a panel of human acute myeloid leukemia and multiple myeloma cell lines with endogenous or ectopic expression of one or more ABC transporter proteins, and using primary leukemia cell samples, we found that PK11195 broadly inhibits ABC transporter function, affecting not only Pgp but also MRP and BCRP. PK11195 blocked efflux often more effectively than CSA in Pgp+, MRP+ and BCRP+ cells, blocked efflux as well as the MRP modulator, MK-571, in MRP+ cells, and inhibited as well or better than the BCRP modulator, GF120918, in BCRP+ cells. Compared to parental cell lines, sublines over-expressing Pgp, MRP, or BCRP showed relative MIT-resistance in cytotoxicity assays. PK11195 co-treatments significantly increased MIT cytotoxicity in such cell lines expressing relatively high levels of one or more clinically relevant drug efflux protein even when anti-apoptotic proteins were also expressed. Ectopic expression models confirmed that pBR can directly mediate chemosensitizing by PK11195, presumably via mitochondrial activities, but that pBR expression is unnecessary for PK11195-mediated efflux inhibition. PK11195 bound plasma membrane sites in Pgp+ cells, stimulated Pgp-associated ATPase activity, and caused conformational changes of Pgp, suggesting that PK11195 modulates Pgp-mediated efflux by direct transporter interaction(s). PK11195 and CSA bound non-competitively in Pgp+ cells, indicating that PK11195 interacts with Pgp at sites that are distinct from CSA-binding sites. Discussion: PK11195 promotes chemotherapy-induced apoptosis by a pBR-dependent mitochondrial mechanism, and broadly blocks drug efflux by a pBR-independent, ABC transporter-dependent mechanism. Since PK11195 concentrations that are effective in vitro can be safely achieved in vivo, PK11195 may be a useful clinical chemosensitizer in patients with MDR+ malignancies.

scholarly journals Myosin V

2004 ◽  
Vol 164 (6) ◽  
pp. 877-886 ◽  
Author(s):  
Dimitry N. Krementsov ◽  
Elena B. Krementsova ◽  
Kathleen M. Trybus
Keyword(s):  
Atpase Activity ◽  
Conformational Change ◽  
Calcium Binding ◽  
Myosin V ◽  
Electron Microscopic ◽  
Microscopic Images ◽  
High Affinity ◽  
Paradoxical Finding ◽  
Hydrodynamic Data ◽  
Compact Conformation

Calcium activates the ATPase activity of tissue-purified myosin V, but not that of shorter expressed constructs. Here, we resolve this discrepancy by comparing an expressed full-length myosin V (dFull) to three shorter constructs. Only dFull has low ATPase activity in EGTA, and significantly higher activity in calcium. Based on hydrodynamic data and electron microscopic images, the inhibited state is due to a compact conformation that is possible only with the whole molecule. The paradoxical finding that dFull moved actin in EGTA suggests that binding of the molecule to the substratum turns it on, perhaps mimicking cargo activation. Calcium slows, but does not stop the rate of actin movement if excess calmodulin (CaM) is present. Without excess CaM, calcium binding to the high affinity sites dissociates CaM and stops motility. We propose that a folded-to-extended conformational change that is controlled by calcium and CaM, and probably by cargo binding itself, regulates myosin V's ability to transport cargo in the cell.

scholarly journals A Novel Synthetic Dihydroindeno[1,2-b] Indole Derivative (LS-2-3j) Reverses ABCB1- and ABCG2-Mediated Multidrug Resistance in Cancer Cells

Molecules ◽  
2018 ◽  
Vol 23 (12) ◽  
pp. 3264 ◽  
Author(s):  
Chao Guo ◽  
Fangyuan Liu ◽  
Jie Qi ◽  
Jiahui Ma ◽  
Shiqi Lin ◽  
...  
Keyword(s):  
Multidrug Resistance ◽  
Protein Expression ◽  
Atpase Activity ◽  
Cancer Cells ◽  
Abc Transporter ◽  
Dependent Manner ◽  
Concentration Dependent Manner ◽  
Expression Levels ◽  
Chemotherapy Drugs ◽  
Diphenyltetrazolium Bromide

10-oxo-5-(3-(pyrrolidin-1-yl) propyl)-5,10-dihydroindeno [1,2-b] indol-9-yl propionate (LS-2-3j) is a new chemically synthesized indole compound and some related analogues are known to be inhibitors (such as alectinib and Ko143) of ATP-binding cassette (ABC) transporters, especially the ABC transporter subfamily B member 1 (ABCB1) and the ABC transporter subfamily G member 2 (ABCG2). This study aimed to evaluate the multidrug resistance (MDR) reversal effects and associated mechanisms of LS-2-3j in drug-resistant cancer cells. The inhibition of cell proliferation in tested agents was evaluated by the 3-(4,5-dimethylthiazol)-2,5-diphenyltetrazolium bromide (MTT) assay. Accumulation or efflux of chemotherapy drugs was analyzed by flow cytometry. The ATPase activity was measured using an ATPase activity assay kit. The mRNA transcripts and protein expression levels were detected by real-time PCR and Western blot, respectively. In this connection, LS-2-3j significantly enhanced the activity of chemotherapeutic drugs in MDR cells and could significantly increase the intracellular accumulation of doxorubicin (DOX) and mitoxantrone (MITX) by inhibiting the function of the efflux pumps in ABCB1- or ABCG2-overexpressing cells. Furthermore, reduced ATPase activity, mRNA transcription, and protein expression levels of ABCB1 and ABCG2 were observed in a concentration dependent manner in MDR cancer cells.

scholarly journals Modulation of ATPase Activity by Physical Disengagement of the ATP-binding Domains of an ABC Transporter, the Histidine Permease

1999 ◽  
Vol 274 (26) ◽  
pp. 18310-18318 ◽  
Author(s):  
Pei-Qi Liu ◽  
Cheng E. Liu ◽  
Giovanna Ferro-Luzzi Ames
Keyword(s):  
Atpase Activity ◽  
Abc Transporter ◽  
Atp Binding ◽  
Binding Domains ◽  
Histidine Permease
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