scholarly journals Structures of ABCG2 under turnover conditions reveal a key step in the drug transport mechanism

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Qin Yu ◽  
Dongchun Ni ◽  
Julia Kowal ◽  
Ioannis Manolaridis ◽  
Scott M. Jackson ◽  
...  
Keyword(s):  
Conformational Changes ◽  
Drug Transport ◽  
Reaction Cycle ◽  
Rate Limiting Step ◽  
Exogenous Substrate ◽  
Endogenous Substrate ◽  
Inhibitory Antibodies ◽  
Rate Limiting ◽  
Drug Pharmacokinetics ◽  
Cytoplasmic Side

AbstractABCG2 is a multidrug transporter that affects drug pharmacokinetics and contributes to multidrug resistance of cancer cells. In previously reported structures, the reaction cycle was halted by the absence of substrates or ATP, mutation of catalytic residues, or the presence of small-molecule inhibitors or inhibitory antibodies. Here we present cryo-EM structures of ABCG2 under turnover conditions containing either the endogenous substrate estrone-3-sulfate or the exogenous substrate topotecan. We find two distinct conformational states in which both the transport substrates and ATP are bound. Whereas the state turnover-1 features more widely separated NBDs and an accessible substrate cavity between the TMDs, turnover-2 features semi-closed NBDs and an almost fully occluded substrate cavity. Substrate size appears to control which turnover state is mainly populated. The conformational changes between turnover-1 and turnover-2 states reveal how ATP binding is linked to the closing of the cytoplasmic side of the TMDs. The transition from turnover-1 to turnover-2 is the likely bottleneck or rate-limiting step of the reaction cycle, where the discrimination of substrates and inhibitors occurs.

scholarly journals Structures of ABCG2 under turnover conditions reveal a key step in drug transport mechanism

2021 ◽  
Author(s):  
Qin Yu ◽  
Dongchun Ni ◽  
Julia Kowal ◽  
Ioannis Manolaridis ◽  
Scott M. Jackson ◽  
...  
Keyword(s):  
Conformational Changes ◽  
Transport Mechanism ◽  
Drug Transport ◽  
Structural Basis ◽  
Reaction Cycle ◽  
Rate Limiting Step ◽  
Exogenous Substrate ◽  
Transport Cycle ◽  
Endogenous Substrate ◽  
Rate Limiting

ABCG2 is a multidrug transporter expressed widely in the human body. Its physiological substrates include steroid derivatives and uric acid. In addition, it extrudes many structurally diverse cytotoxic drugs from various cells, thus affecting drug pharmacokinetics and contributing to multidrug resistance of cancer cells. Previous studies have revealed structures of ABCG2 bound to transport substrates, nucleotides, small-molecule inhibitors and inhibitory antibodies. However, the transport mechanism is not well-understood because all previous structures described trapped states, where the reaction cycle was halted by the absence of substrates or ATP, mutation of catalytic residues, or the presence of inhibitors. Here we present cryo-EM structures of nanodisc-reconstituted human ABCG2 under turnover conditions containing either the endogenous substrate estrone-3-sulfate or the exogenous substrate topotecan. We found two distinct conformational states in which both the transport substrates and ATP are bound. Whereas the state turnover-1 features more widely separated NBDs and an accessible cavity between the TMDs, turnover-2 features semi-closed NBDs and an almost fully occluded cavity between the TMDs. The transition from turnover-1 to turnover-2 includes conformational changes that link the binding of ATP by the NBDs to the closing of the cytoplasmic side of the TMDs. The size of the substrate appears to control which turnover state corresponds to the main state in the transport cycle. The transition from turnover-1 to turnover-2 is the likely bottleneck or rate-limiting step of the reaction cycle, where the discrimination of substrates and inhibitors occurs. Our results provide a structural basis of substrate specificity of ABCG2 and provide key insight to understand the transport cycle.

scholarly journals A mechanism for acetylcholine receptor gating based on structure, coupling, phi, and flip

2016 ◽  
Vol 149 (1) ◽  
pp. 85-103 ◽  
Author(s):  
Shaweta Gupta ◽  
Srirupa Chakraborty ◽  
Ridhima Vij ◽  
Anthony Auerbach
Keyword(s):  
Conformational Changes ◽  
Nicotinic Acetylcholine Receptors ◽  
Acetylcholine Receptors ◽  
Single Channel ◽  
Bubble Formation ◽  
Transmembrane Helix ◽  
Energy Landscapes ◽  
Rate Limiting Step ◽  
Sequence Domain ◽  
Rate Limiting

Nicotinic acetylcholine receptors are allosteric proteins that generate membrane currents by isomerizing (“gating”) between resting and active conformations under the influence of neurotransmitters. Here, to explore the mechanisms that link the transmitter-binding sites (TBSs) with the distant gate, we use mutant cycle analyses to measure coupling between residue pairs, phi value analyses to sequence domain rearrangements, and current simulations to reproduce a microsecond shut component (“flip”) apparent in single-channel recordings. Significant interactions between amino acids separated by >15 Å are rare; an exception is between the αM2–M3 linkers and the TBSs that are ∼30 Å apart. Linker residues also make significant, local interactions within and between subunits. Phi value analyses indicate that without agonists, the linker is the first region in the protein to reach the gating transition state. Together, the phi pattern and flip component suggest that a complete, resting↔active allosteric transition involves passage through four brief intermediate states, with brief shut events arising from sojourns in all or a subset. We derive energy landscapes for gating with and without agonists, and propose a structure-based model in which resting→active starts with spontaneous rearrangements of the M2–M3 linkers and TBSs. These conformational changes stabilize a twisted extracellular domain to promote transmembrane helix tilting, gate dilation, and the formation of a “bubble” that collapses to initiate ion conduction. The energy landscapes suggest that twisting is the most energetically unfavorable step in the resting→active conformational change and that the rate-limiting step in the reverse process is bubble formation.

scholarly journals Conformational Changes Represent the Rate-Limiting Step in the Transport Cycle of Maize SUCROSE TRANSPORTER1

2013 ◽  
Vol 25 (8) ◽  
pp. 3010-3021 ◽  
Author(s):  
Carmen Derrer ◽  
Anke Wittek ◽  
Ernst Bamberg ◽  
Armando Carpaneto ◽  
Ingo Dreyer ◽  
...  
Keyword(s):  
Conformational Changes ◽  
Rate Limiting Step ◽  
Limiting Step ◽  
Transport Cycle ◽  
Rate Limiting

Structure-function relationships in an anion-translocating ATPase

2000 ◽  
Vol 28 (4) ◽  
pp. 520-526
Author(s):  
H. Bhattacharjee ◽  
T. Zhou ◽  
J. Li ◽  
D. L. Gatti ◽  
A. R. Walmsley ◽  
...  
Keyword(s):  
Conformational Changes ◽  
Abc Transporters ◽  
Efflux Pump ◽  
Allosteric Activation ◽  
Ancestral Gene ◽  
Binding Domains ◽  
Fluorescence Measurements ◽  
Rate Limiting Step ◽  
Limiting Step ◽  
Rate Limiting

The ArsAB ATPase is an efflux pump located in the inner membrane of Escherichia coli. This transport ATPase confers resistance to arsenite and antimonite by their extrusion from the cells. The pump is composed of two subunits, the catalytic ArsA subunit and the membrane subunit ArsB. The complex is similar in many ways to ATP-binding cassette (‘ABC’) transporters, which typically have two groups of six transmembrane-spanning helical segments and two nucleotide-binding domains (NBDs). The 45 kDa ArsB protein has 12 transmembrane-spanning segments. ArsB contains the substrate translocation pathway and is capable of functioning as an anion uniporter. The 63 kDa ArsA protein is a substrate-activated ATPase. It has two homologous halves, A1 and A2, which are clearly the result of an ancestral gene duplication and fusion. Each half has a consensus NBD. The mechanism of allosteric activation of the ArsA ATPase has been elucidated by a combination of molecular genetics and biochemical, structural and kinetic analyses. Conformational changes produced by binding of substrates, activator and/or products could be revealed by stopped-flow fluorescence measurements with single-tryptophan derivatives of ArsA. The results demonstrate that the rate-limiting step in the overall reaction is a slow isomerization between two conformations of the enzyme. Allosteric activation increases the rate of this isomerization such that product release becomes rate-limiting, thus accelerating catalysis. ABC transporters, which exhibit similar substrate activation of ATPase activity, can undergo similar conformational changes to overcome a rate-limiting step. Thus the ArsAB pump is a useful model for elucidating mechanistic aspects of the ABC superfamily of transport ATPases.

Structural analysis of cofactor binding for a prolyl 4-hydroxylase from the pathogenic bacteriumBacillus anthracis

2016 ◽  
Vol 72 (5) ◽  
pp. 675-681 ◽  
Author(s):  
Nicholas J. Schnicker ◽  
Mishtu Dey
Keyword(s):  
Conformational Changes ◽  
Active Site ◽  
Metal Ion ◽  
Helical Structure ◽  
Future Design ◽  
Cofactor Binding ◽  
Nonheme Iron Enzymes ◽  
Rate Limiting Step ◽  
Fibrotic Disorders ◽  
Rate Limiting

The prolyl 4-hydroxylases (P4Hs) are mononuclear nonheme iron enzymes that catalyze the formation of 4R-hydroxyproline from many different substrates, with various biological implications. P4H is a key player in collagen accumulation, which has implications in fibrotic disorders. The stabilization of collagen triple-helical structureviaprolyl hydroxylation is the rate-limiting step in collagen biosynthesis, and therefore P4H has been extensively investigated as a potential therapeutic target of fibrotic disease. Understanding how these enzymes recognize cofactors and substrates is important and will aid in the future design of inhibitors of P4H. In this article, X-ray crystal structures of a metallocofactor- and α-ketoglutarate (αKG)-bound form of P4H fromBacillus anthracis(BaP4H) are reported. Structures of BaP4H were solved at 1.63 and 2.35 Å resolution and contained a cadmium ion and αKG bound in the active site. The αKG–Cd–BaP4H ternary complex reveals conformational changes of conserved residues upon the binding of metal ion and αKG, resulting in a closed active-site configuration required for dioxygen, substrate binding and catalysis.

scholarly journals Characterization of the kinetic cycle of an ABC transporter by single-molecule and cryo-EM analyses

eLife ◽  
2020 ◽  
Vol 9 ◽  
Author(s):  
Ling Wang ◽  
Zachary Lee Johnson ◽  
Michael R Wasserman ◽  
Jesper Levring ◽  
Jue Chen ◽  
...  
Keyword(s):  
Single Molecule ◽  
Conformational Changes ◽  
Atp Hydrolysis ◽  
Chemotherapeutic Agents ◽  
Multidrug Resistance Protein 1 ◽  
Rate Limiting Step ◽  
Single Molecule Fluorescence Spectroscopy ◽  
Resistance Protein ◽  
Rate Limiting

ATP-binding cassette (ABC) transporters are molecular pumps ubiquitous across all kingdoms of life. While their structures have been widely reported, the kinetics governing their transport cycles remain largely unexplored. Multidrug resistance protein 1 (MRP1) is an ABC exporter that extrudes a variety of chemotherapeutic agents and native substrates. Previously, the structures of MRP1 were determined in an inward-facing (IF) or outward-facing (OF) conformation. Here, we used single-molecule fluorescence spectroscopy to track the conformational changes of bovine MRP1 (bMRP1) in real time. We also determined the structure of bMRP1 under active turnover conditions. Our results show that substrate stimulates ATP hydrolysis by accelerating the IF-to-OF transition. The rate-limiting step of the transport cycle is the dissociation of the nucleotide-binding-domain dimer, while ATP hydrolysis per se does not reset MRP1 to the resting state. The combination of structural and kinetic data illustrates how different conformations of MRP1 are temporally linked and how substrate and ATP alter protein dynamics to achieve active transport.

scholarly journals Multiple intermediate states precede pore block during N-type inactivation of a voltage-gated potassium channel

2009 ◽  
Vol 134 (1) ◽  
pp. 15-34 ◽  
Author(s):  
Alison Prince-Carter ◽  
Paul J. Pfaffinger
Keyword(s):  
Conformational Changes ◽  
Polar Region ◽  
Single Step ◽  
Side Window ◽  
Voltage Gated ◽  
Rate Limiting Step ◽  
Limiting Step ◽  
Intermediate States ◽  
N Terminus ◽  
Rate Limiting

N-type inactivation of voltage-gated potassium channels is an autoinhibitory process that occurs when the N terminus binds within the channel pore and blocks conduction. N-type inactivation and recovery occur with single-exponential kinetics, consistent with a single-step reaction where binding and block occur simultaneously. However, recent structure–function studies have suggested the presence of a preinactivated state whose formation and loss regulate inactivation and recovery kinetics. Our studies on N-type inactivation of the Shaker-type AKv1 channel support a multiple-step inactivation process involving a series of conformational changes in distinct regions of the N terminus that we have named the polar, flex, and latch regions. The highly charged polar region forms interactions with the surface of the channel leading up to the side window openings between the T1 domain and the channel transmembrane domains, before the rate-limiting step occurs. This binding culminates with a specific electrostatic interaction between R18 and EDE161-163 located at the entrance to the side windows. The latch region appears to work together with the flex region to block the pore after polar region binding occurs. Analysis of tail currents for a latch region mutant shows that both blocked and unblocked states exist after the rate-limiting transition is passed. Our results suggest that at least two intermediate states exist for N-type inactivation: a polar region–bound state that is formed before the rate-limiting step, and a pre-block state that is formed by the flex and latch regions during the rate-limiting step.

Ornithine Decarboxylase Activity in Cultured Endothelial Cells Stimulated by Serum, Thrombin and Serotonin

1978 ◽  
Vol 39 (02) ◽  
pp. 496-503 ◽  
Author(s):  
P A D’Amore ◽  
H B Hechtman ◽  
D Shepro
Keyword(s):  
Endothelial Cells ◽  
Ornithine Decarboxylase ◽  
Decarboxylase Activity ◽  
Aortic Endothelial Cells ◽  
Ornithine Decarboxylase Activity ◽  
Rate Limiting Step ◽  
Bovine Aortic Endothelial Cells ◽  
Limiting Step ◽  
Rate Limiting ◽  
Serum Serotonin

SummaryOrnithine decarboxylase (ODC) activity, the rate-limiting step in the synthesis of polyamines, can be demonstrated in cultured, bovine, aortic endothelial cells (EC). Serum, serotonin and thrombin produce a rise in ODC activity. The serotonin-induced ODC activity is significantly blocked by imipramine (10-5 M) or Lilly 11 0140 (10-6M). Preincubation of EC with these blockers together almost completely depresses the 5-HT-stimulated ODC activity. These observations suggest a manner by which platelets may maintain EC structural and metabolic soundness.

Sign in / Sign up

Export Citation Format

Share Document