scholarly journals ATP hydrolysis and nucleotide exit enhance maltose translocation in the MalFGK2E importer

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Bárbara Abreu ◽  
Carlos Cruz ◽  
A. Sofia F. Oliveira ◽  
Cláudio M. Soares
Keyword(s):  
Conformational Changes ◽  
Abc Transporters ◽  
Atp Hydrolysis ◽  
Binding Pocket ◽  
Potential Of Mean Force ◽  
Type I ◽  
Concomitant Increase ◽  
Transport Cycle ◽  
Dynamics Simulations ◽  
Key Residues

AbstractATP binding cassette (ABC) transporters employ ATP hydrolysis to harness substrate translocation across membranes. The Escherichia coli MalFGK2E maltose importer is an example of a type I ABC importer and a model system for this class of ABC transporters. The MalFGK2E importer is responsible for the intake of malto-oligossacharides in E.coli. Despite being extensively studied, little is known about the effect of ATP hydrolysis and nucleotide exit on substrate transport. In this work, we studied this phenomenon using extensive molecular dynamics simulations (MD) along with potential of mean force calculations of maltose transport across the pore, in the pre-hydrolysis, post-hydrolysis and nucleotide-free states. We concluded that ATP hydrolysis and nucleotide exit trigger conformational changes that result in the decrease of energetic barriers to maltose translocation towards the cytoplasm, with a concomitant increase of the energy barrier in the periplasmic side of the pore, contributing for the irreversibility of the process. We also identified key residues that aid in positioning and orientation of maltose, as well as a novel binding pocket for maltose in MalG. Additionally, ATP hydrolysis leads to conformations similar to the nucleotide-free state. This study shows the contribution of ATP hydrolysis and nucleotide exit in the transport cycle, shedding light on ABC type I importer mechanisms.

scholarly journals Cryo-EM structure of an elusive pre-transport intermediate of the multidrug transporter BmrCD reveals modes of asymmetric drug binding

2021 ◽  
Author(s):  
Tarjani M Thaker ◽  
Smriti Mishra ◽  
Wenchang Zhou ◽  
Jose Faraldo-Gomez ◽  
Hassane Mchaourab ◽  
...  
Keyword(s):  
Conformational Changes ◽  
Abc Transporters ◽  
Rational Design ◽  
Transmembrane Domain ◽  
Atp Hydrolysis ◽  
Domain Architecture ◽  
Drug Binding ◽  
Design And Optimization ◽  
Dynamics Simulations ◽  
Double Electron Electron Resonance

Vectorial substrate efflux by ATP binding cassette (ABC) transporters, which play a major role in multidrug resistance, entails the ATP-powered interconversion of the transporter between stable intermediates. Despite recent progress in structure elucidation of ABC transporters, a number of such intermediates have yet to be visualized and mechanistically interpreted. Here, we combine single particle cryo-EM, Double Electron Electron Resonance (DEER) spectroscopy with Molecular Dynamics simulations to profile and mechanistically frame the conformation of a hitherto unobserved intermediate in the context of BmrCD, a heterodimeric multidrug ABC exporter from Bacillus subtilis. In our cryo-EM structure, BmrCD adopts an inward facing architecture bound to both ATP and the substrate Hoechst-33342 and is capped by an extracellular domain which undergoes ATP-dependent conformational changes. A striking feature of the structure is a symmetric arrangement of the nucleotide-binding domain (NBD) in the presence of ATP whereas binding of Hoechst at two distinct sites in an acidic pocket stabilizes an asymmetric arrangement of the transmembrane domain architecture (TMD). Mutation of residues coordinating Hoechst in the structure abrogates the cooperative stimulation of ATP hydrolysis. In conjunction with previous studies, our findings suggest a mechanistic role for symmetry mismatch between NBDs and TMDs in the conformational cycle of ABC transporters. Moreover, the resolved structures of bimodally-bound drugs are of notable importance for future rational design and optimization of molecules for targeted transport inhibition of ABC transporters.

Molecular Basis of Glucose Transport Mechanism in Plants

2019 ◽  
Author(s):  
Balaji Selvam ◽  
Ya-Chi Yu ◽  
Liqing Chen ◽  
Diwakar Shukla
Keyword(s):  
Molecular Dynamics ◽  
Free Energy ◽  
Molecular Dynamics Simulations ◽  
Conformational Changes ◽  
Transport Mechanism ◽  
Conformational Dynamics ◽  
Site Directed Mutagenesis ◽  
Free Energy Barrier ◽  
Transport Cycle ◽  
Dynamics Simulations

<p>The SWEET family belongs to a class of transporters in plants that undergoes large conformational changes to facilitate transport of sugar molecules across the cell membrane. However, the structures of their functionally relevant conformational states in the transport cycle have not been reported. In this study, we have characterized the conformational dynamics and complete transport cycle of glucose in OsSWEET2b transporter using extensive molecular dynamics simulations. Using Markov state models, we estimated the free energy barrier associated with different states as well as 1 for the glucose the transport mechanism. SWEETs undergoes structural transition to outward-facing (OF), Occluded (OC) and inward-facing (IF) and strongly support alternate access transport mechanism. The glucose diffuses freely from outside to inside the cell without causing major conformational changes which means that the conformations of glucose unbound and bound snapshots are exactly same for OF, OC and IF states. We identified a network of hydrophobic core residues at the center of the transporter that restricts the glucose entry to the cytoplasmic side and act as an intracellular hydrophobic gate. The mechanistic predictions from molecular dynamics simulations are validated using site-directed mutagenesis experiments. Our simulation also revealed hourglass like intermediate states making the pore radius narrower at the center. This work provides new fundamental insights into how substrate-transporter interactions actively change the free energy landscape of the transport cycle to facilitate enhanced transport activity.</p>

A molecular understanding of the catalytic cycle of the nucleotide-binding domain of the ABC transporter HlyB

2005 ◽  
Vol 33 (5) ◽  
pp. 990-995 ◽  
Author(s):  
J. Zaitseva ◽  
S. Jenewein ◽  
C. Oswald ◽  
T. Jumpertz ◽  
I.B. Holland ◽  
...  
Keyword(s):  
Abc Transporter ◽  
Conformational Changes ◽  
Structural Changes ◽  
Atp Hydrolysis ◽  
Central Element ◽  
Nucleotide Binding ◽  
Single Step ◽  
Catalytic Cycle ◽  
Type I ◽  
Individual Crystal

The ABC transporter (ATP-binding-cassette transporter) HlyB (haemolysin B) is the central element of a type I secretion machinery, dedicated to the secretion of the toxin HlyA in Escherichia coli. In addition to the ABC transporter, two other indispensable elements are necessary for the secretion of the toxin across two membranes in a single step: the transenvelope protein HlyD and the outer membrane protein TolC. Despite the fact that the hydrolysis of ATP by HlyB fuels secretion of HlyA, the essential features of the underlying transport mechanism remain an enigma. Similar to all other ABC transporters, ranging from bacteria to man, HlyB is composed of two NBDs (nucleotide-binding domains) and two transmembrane domains. Here we summarize our detailed biochemical, biophysical and structural studies aimed at an understanding of the molecular principles of how ATP-hydrolysis is coupled to energy transduction, including the conformational changes occurring during the catalytic cycle, leading to substrate transport. We have obtained individual crystal structures for each single ground state of the catalytic cycle. From these and other biochemical and mutational studies, we shall provide a detailed molecular picture of the steps governing intramolecular communication and the utilization of chemical energy, due to ATP hydrolysis, in relation to resulting structural changes within the NBD. These data will be summarized in a general model to explain how these molecular machines achieve translocation of molecules across biological membranes.

scholarly journals RNA Packaging Motor: From Structure to Quantum Mechanical Modelling and Sequential-Stochastic Mechanism

2008 ◽  
Vol 9 (3-4) ◽  
pp. 351-369 ◽  
Author(s):  
Jelena Telenius ◽  
Anders E. Wallin ◽  
Michal Straka ◽  
Hongbo Zhang ◽  
Erika J. Mancini ◽  
...  
Keyword(s):  
Conformational Changes ◽  
Atp Hydrolysis ◽  
Structural Information ◽  
Molecular Motor ◽  
Structural Similarity ◽  
Binding Pocket ◽  
Reaction Coordinate ◽  
Rna Packaging ◽  
Cooperative Action ◽  
Empty Capsid

The bacteriophages of theCystoviridaefamily package their single stranded RNA genomic precursors into empty capsid (procapsids) using a hexameric packaging ATPase motor (P4). This molecular motor shares sequence and structural similarity with RecA-like hexameric helicases. A concerted structural, mutational and kinetic analysis helped to define the mechanical reaction coordinate,i.e.the conformational changes associated with RNA translocation. The results also allowed us to propose a possible scheme of coupling between ATP hydrolysis and translocation which requires the cooperative action of three consecutive subunits. Here, we first test this model by preparing hexamers with defined proportions of wild type and mutant subunits and measuring their activity. Then, we develop a stochastic kinetic model which accounts for the catalytic cooperativity of the P4 hexamer. Finally, we use the available structural information to construct a quantum-chemical model of the chemical reaction coordinate and obtain a detailed description of the electron density changes during ATP hydrolysis. The model explains the results of the mutational analyses and yields new insights into the role of several conserved residues within the ATP binding pocket. These hypotheses will guide future experimental work.

scholarly journals In vitro study of Hesperetin and Hesperidin as inhibitors of zika and chikungunya virus proteases

PLoS ONE ◽  
2021 ◽  
Vol 16 (3) ◽  
pp. e0246319
Author(s):  
Raphael J. Eberle ◽  
Danilo S. Olivier ◽  
Carolina C. Pacca ◽  
Clarita M. S. Avilla ◽  
Mauricio L. Nogueira ◽  
...  
Keyword(s):  
Conformational Changes ◽  
Chikungunya Virus ◽  
In Vitro Study ◽  
3D Models ◽  
Binding Pocket ◽  
Starting Point ◽  
Low Cytotoxicity ◽  
Inhibitory Potential ◽  
Dynamics Simulations

The potential outcome of flavivirus and alphavirus co-infections is worrisome due to the development of severe diseases. Hundreds of millions of people worldwide live under the risk of infections caused by viruses like chikungunya virus (CHIKV, genus Alphavirus), dengue virus (DENV, genus Flavivirus), and zika virus (ZIKV, genus Flavivirus). So far, neither any drug exists against the infection by a single virus, nor against co-infection. The results described in our study demonstrate the inhibitory potential of two flavonoids derived from citrus plants: Hesperetin (HST) against NS2B/NS3pro of ZIKV and nsP2pro of CHIKV and, Hesperidin (HSD) against nsP2pro of CHIKV. The flavonoids are noncompetitive inhibitors and the determined IC50 values are in low µM range for HST against ZIKV NS2B/NS3pro (12.6 ± 1.3 µM) and against CHIKV nsP2pro (2.5 ± 0.4 µM). The IC50 for HSD against CHIKV nsP2pro was 7.1 ± 1.1 µM. The calculated ligand efficiencies for HST were > 0.3, which reflect its potential to be used as a lead compound. Docking and molecular dynamics simulations display the effect of HST and HSD on the protease 3D models of CHIKV and ZIKV. Conformational changes after ligand binding and their effect on the substrate-binding pocket of the proteases were investigated. Additionally, MTT assays demonstrated a very low cytotoxicity of both the molecules. Based on our results, we assume that HST comprise a chemical structure that serves as a starting point molecule to develop a potent inhibitor to combat CHIKV and ZIKV co-infections by inhibiting the virus proteases.

Molecular Basis of Glucose Transport Mechanism in Plants

2019 ◽  
Author(s):  
Balaji Selvam ◽  
Ya-Chi Yu ◽  
Liqing Chen ◽  
Diwakar Shukla
Keyword(s):  
Molecular Dynamics ◽  
Free Energy ◽  
Molecular Dynamics Simulations ◽  
Conformational Changes ◽  
Transport Mechanism ◽  
Conformational Dynamics ◽  
Site Directed Mutagenesis ◽  
Free Energy Barrier ◽  
Transport Cycle ◽  
Dynamics Simulations

<p>The SWEET family belongs to a class of transporters in plants that undergoes large conformational changes to facilitate transport of sugar molecules across the cell membrane. However, the structures of their functionally relevant conformational states in the transport cycle have not been reported. In this study, we have characterized the conformational dynamics and complete transport cycle of glucose in OsSWEET2b transporter using extensive molecular dynamics simulations. Using Markov state models, we estimated the free energy barrier associated with different states as well as 1 for the glucose the transport mechanism. SWEETs undergoes structural transition to outward-facing (OF), Occluded (OC) and inward-facing (IF) and strongly support alternate access transport mechanism. The glucose diffuses freely from outside to inside the cell without causing major conformational changes which means that the conformations of glucose unbound and bound snapshots are exactly same for OF, OC and IF states. We identified a network of hydrophobic core residues at the center of the transporter that restricts the glucose entry to the cytoplasmic side and act as an intracellular hydrophobic gate. The mechanistic predictions from molecular dynamics simulations are validated using site-directed mutagenesis experiments. Our simulation also revealed hourglass like intermediate states making the pore radius narrower at the center. This work provides new fundamental insights into how substrate-transporter interactions actively change the free energy landscape of the transport cycle to facilitate enhanced transport activity.</p>

Chloride-dependent conformational changes in the GlyT1 glycine transporter

2020 ◽  
Author(s):  
Yuan-Wei Zhang ◽  
Stacy Uchendu ◽  
Vanessa Leone ◽  
Richard T. Bradshaw ◽  
Ntumba Sangwa ◽  
...  
Keyword(s):  
Conformational Changes ◽  
Ion Pair ◽  
Cysteine Residues ◽  
Glycine Transporter ◽  
Open Conformation ◽  
Transporter Family ◽  
Transport Cycle ◽  
Dynamics Simulations ◽  
Insight Into

AbstractThe human GlyT1 glycine transporter requires chloride for its function. However, the mechanism by which Cl- exerts its influence is unknown. To examine the role that Cl- plays in the transport cycle, we measured the effect of Cl- on both glycine binding and conformational changes. The ability of glycine to displace the high-affinity radioligand [3H]CHIBA-3007 required Na+ and was potentiated over 1000-fold by Cl-. We generated GlyT1b mutants containing reactive cysteine residues in either the extracellular or cytoplasmic permeation pathways and measured changes in the reactivity of those cysteine residues as indicators of conformational changes in response to ions and substrate. Na+ increased accessibility in the extracellular pathway and decreased it in the cytoplasmic pathway, consistent with stabilizing an outward-open conformation as observed in other members of this transporter family. In the presence of Na+, both glycine and Cl- independently shifted the conformation of GlyT1b toward an outward-closed conformation. Together, Na+, glycine and Cl- stabilized an inward-open conformation of GlyT1b. We then examined whether Cl- acts by interacting with a conserved glutamine to allow formation of an ion pair that stabilizes the closed state of the extracellular pathway. Molecular dynamics simulations of a GlyT1 homologue indicated that this ion pair is formed more frequently as that pathway closes. Mutation of the glutamine blocked the effect of Cl-, and substituting it with glutamate or lysine resulted in outward- or inward-facing transporter conformations, respectively. These results provide novel and unexpected insight into the role of Cl- in this family of transporters.

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.

scholarly journals Computational Insights into Allosteric Conformational Modulation of P-Glycoprotein by Substrate and Inhibitor Binding

Molecules ◽  
2020 ◽  
Vol 25 (24) ◽  
pp. 6006
Author(s):  
Juan Xing ◽  
Shuheng Huang ◽  
Yu Heng ◽  
Hu Mei ◽  
Xianchao Pan
Keyword(s):  
Conformational Changes ◽  
Atp Hydrolysis ◽  
Molecular Design ◽  
Conformational Dynamics ◽  
Water Environment ◽  
Inhibitor Binding ◽  
Binding Domains ◽  
Allosteric Communication ◽  
P Glycoprotein ◽  
Dynamics Simulations

The ATP-binding cassette (ABC) transporter P-glycoprotein (P-gp) is a physiologically essential membrane protein that protects many tissues against xenobiotic molecules, but limits the access of chemotherapeutics into tumor cells, thus contributing to multidrug resistance. The atomic-level mechanism of how substrates and inhibitors differentially affect the ATP hydrolysis by P-gp remains to be elucidated. In this work, atomistic molecular dynamics simulations in an explicit membrane/water environment were performed to explore the effects of substrate and inhibitor binding on the conformational dynamics of P-gp. Distinct differences in conformational changes that mainly occurred in the nucleotide-binding domains (NBDs) were observed from the substrate- and inhibitor-bound simulations. The binding of rhodamine-123 can increase the probability of the formation of an intermediate conformation, in which the NBDs were closer and better aligned, suggesting that substrate binding may prime the transporter for ATP hydrolysis. By contrast, the inhibitor QZ-Leu stabilized NBDs in a much more separated and misaligned conformation, which may result in the deficiency of ATP hydrolysis. The significant differences in conformational modulation of P-gp by substrate and inhibitor binding provided a molecular explanation of how these small molecules exert opposite effects on the ATPase activity. A further structural analysis suggested that the allosteric communication between transmembrane domains (TMDs) and NBDs was primarily mediated by two intracellular coupling helices. Our computational simulations provide not only valuable insights into the transport mechanism of P-gp substrates, but also for the molecular design of P-gp inhibitors.

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