scholarly journals The extracellular gate shapes the energy profile of an ABC exporter

2018 ◽  
Author(s):  
Cedric A.J. Hutter ◽  
M. Hadi Timachi ◽  
Lea M. Hürlimann ◽  
Iwan Zimmermann ◽  
Pascal Egloff ◽  
...  
Keyword(s):  
Atpase Activity ◽  
Drug Transport ◽  
Atp Hydrolysis ◽  
Underlying Mechanism ◽  
Single Domain Antibody ◽  
Gate Opening ◽  
Transport Cycle ◽  
Double Electron ◽  
Double Electron Electron Resonance ◽  
Key Steps

ABSTRACTABC exporters harness the energy of ATP to pump substrates across membranes. Extracellular gate opening and closure are key steps of the transport cycle, but the underlying mechanism is poorly understood. Here, we generated a synthetic single domain antibody (sybody) that recognizes the heterodimeric ABC exporter TM287/288 exclusively in the presence of ATP, which was essential to solve a 3.2 Å crystal structure of the outward-facing transporter. The sybody binds to an extracellular wing and strongly inhibits ATPase activity by shifting the transporter’s conformational equilibrium towards the outward-facing state, as shown by double electron-electron resonance (DEER). Mutations that facilitate extracellular gate opening resulted in a comparable equilibrium shift and strongly reduced ATPase activity and drug transport. Using the sybody as conformational probe, we demonstrate that efficient extracellular gate closure is required to dissociate the NBD dimer after ATP hydrolysis to reset the transporter back to its inward-facing state.

scholarly journals MDA5 disease variant M854K prevents ATP-dependent structural discrimination of viral and cellular RNA

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Qin Yu ◽  
Alba Herrero del Valle ◽  
Rahul Singh ◽  
Yorgo Modis
Keyword(s):  
Atpase Activity ◽  
Atp Hydrolysis ◽  
Innate Immune Responses ◽  
Interferon Signaling ◽  
Double Stranded Rna ◽  
Dsrna Binding ◽  
Exogenous Rna ◽  
Health And Disease ◽  
Disease Variant ◽  
Key Steps

AbstractOur innate immune responses to viral RNA are vital defenses. Long cytosolic double-stranded RNA (dsRNA) is recognized by MDA5. The ATPase activity of MDA5 contributes to its dsRNA binding selectivity. Mutations that reduce RNA selectivity can cause autoinflammatory disease. Here, we show how the disease-associated MDA5 variant M854K perturbs MDA5-dsRNA recognition. M854K MDA5 constitutively activates interferon signaling in the absence of exogenous RNA. M854K MDA5 lacks ATPase activity and binds more stably to synthetic Alu:Alu dsRNA. CryoEM structures of MDA5-dsRNA filaments at different stages of ATP hydrolysis show that the K854 sidechain forms polar bonds that constrain the conformation of MDA5 subdomains, disrupting key steps in the ATPase cycle- RNA footprint expansion and helical twist modulation. The M854K mutation inhibits ATP-dependent RNA proofreading via an allosteric mechanism, allowing MDA5 to form signaling complexes on endogenous RNAs. This work provides insights on how MDA5 recognizes dsRNA in health and disease.

scholarly journals Protonation-dependent conformational dynamics of the multidrug transporter EmrE

2016 ◽  
Vol 113 (5) ◽  
pp. 1220-1225 ◽  
Author(s):  
Reza Dastvan ◽  
Axel W. Fischer ◽  
Smriti Mishra ◽  
Jens Meiler ◽  
Hassane S. Mchaourab
Keyword(s):  
Conformational Changes ◽  
Transport Model ◽  
Conformational Dynamics ◽  
Bound State ◽  
Multidrug Transporter ◽  
Transmembrane Helices ◽  
Transport Cycle ◽  
Double Electron ◽  
Double Electron Electron Resonance ◽  
Alternating Access

The small multidrug transporter from Escherichia coli, EmrE, couples the energetically uphill extrusion of hydrophobic cations out of the cell to the transport of two protons down their electrochemical gradient. Although principal mechanistic elements of proton/substrate antiport have been described, the structural record is limited to the conformation of the substrate-bound state, which has been shown to undergo isoenergetic alternating access. A central but missing link in the structure/mechanism relationship is a description of the proton-bound state, which is an obligatory intermediate in the transport cycle. Here we report a systematic spin labeling and double electron electron resonance (DEER) study that uncovers the conformational changes of EmrE subsequent to protonation of critical acidic residues in the context of a global description of ligand-induced structural rearrangements. We find that protonation of E14 leads to extensive rotation and tilt of transmembrane helices 1–3 in conjunction with repacking of loops, conformational changes that alter the coordination of the bound substrate and modulate its access to the binding site from the lipid bilayer. The transport model that emerges from our data posits a proton-bound, but occluded, resting state. Substrate binding from the inner leaflet of the bilayer releases the protons and triggers alternating access between inward- and outward-facing conformations of the substrate-loaded transporter, thus enabling antiport without dissipation of the proton gradient.

scholarly journals MDA5 autoimmune disease variant M854K prevents ATP-dependent structural discrimination of viral and cellular RNA

2021 ◽  
Author(s):  
Qin Yu ◽  
Alba Herrero del Valle ◽  
Rahul Singh ◽  
Yorgo Modis
Keyword(s):  
Autoimmune Disease ◽  
Atpase Activity ◽  
Atp Hydrolysis ◽  
Side Chain ◽  
Innate Immune Responses ◽  
Dsrna Binding ◽  
Exogenous Rna ◽  
Health And Disease ◽  
Disease Variant ◽  
Key Steps

AbstractOur innate immune responses to viral RNA are vital defenses. Long cytosolic double-stranded RNA (dsRNA) is recognized by MDA5. The ATPase activity of MDA5 contributes to its dsRNA binding selectivity. Mutations that reduce RNA selectivity can cause autoimmune disease. Here, we show how the disease-associated MDA5 variant M854K perturbs MDA5-dsRNA recognition. M854K MDA5 constitutively activates interferon signaling in the absence of exogenous RNA. M854K MDA5 lacks ATPase activity and binds more tightly to synthetic Alu:Alu dsRNA. CryoEM structures MDA5-dsRNA filaments at different stages of ATP hydrolysis show that the K854 side-chain forms polar bonds that constrain the conformation of MDA5 subdomains, disrupting key steps in the ATPase cycle-RNA footprint expansion and helical twist modulation. The M854K mutation inhibits ATP-dependent RNA proofreading via a novel allosteric mechanism, allowing MDA5 to form signaling complexes on endogenous RNAs. This work provides new insights on how MDA5 recognizes dsRNA in health and disease.

scholarly journals Disruption of the Unique ABCG-Family NBD:NBD Interface Impacts Both Drug Transport and ATP Hydrolysis

2020 ◽  
Vol 21 (3) ◽  
pp. 759
Author(s):  
Parth Kapoor ◽  
Deborah A. Briggs ◽  
Megan H. Cox ◽  
Ian D. Kerr
Keyword(s):  
Atpase Activity ◽  
Drug Transport ◽  
Atp Hydrolysis ◽  
Contact Region ◽  
Chemotherapy Resistance ◽  
Dimer Interface ◽  
Binding Domains ◽  
Related Family ◽  
Allosteric Communication ◽  
C Terminus

ABCG2 is one of a triumvirate of human multidrug ATP binding cassette (ABC) transporters that are implicated in the defense of cells and tissues against cytotoxic chemicals, but these transporters can also confer chemotherapy resistance states in oncology. Understanding the mechanism of ABCG2 is thus imperative if we are to be able to counter its deleterious activity. The structure of ABCG2 and its related family members (ABCG5/G8) demonstrated that there were two interfaces between the nucleotide binding domains (NBD). In addition to the canonical ATP “sandwich-dimer” interface, there was a second contact region between residues at the C-terminus of the NBD. We investigated this second interface by making mutations to a series of residues that are in close interaction with the opposite NBD. Mutated ABCG2 isoforms were expressed in human embryonic kidney (HEK) 293T cells and analysed for targeting to the membrane, drug transport, and ATPase activity. Mutations to this second interface had a number of effects on ABCG2, including altered drug specificity, altered drug transport, and, in two mutants, a loss of ATPase activity. The results demonstrate that this region is particularly sensitive to mutation and can impact not only direct, local NBD events (i.e., ATP hydrolysis) but also the allosteric communication to the transmembrane domains and drug transport.

Direct Observation of Chain Lengths and Conformations in Oligofluorene Distributions from Controlled Polymerization by Double Electron-Electron Resonance

2019 ◽  
Author(s):  
Dennis Bücker ◽  
Annika Sickinger ◽  
Julian D. Ruiz Perez ◽  
Manuel Oestringer ◽  
Stefan Mecking ◽  
...  
Keyword(s):  
Chain Length ◽  
Length Distribution ◽  
Catalyst System ◽  
Chain Conformation ◽  
Controlled Polymerization ◽  
Chain Length Distribution ◽  
Electron Resonance ◽  
Double Electron ◽  
The Individual ◽  
Double Electron Electron Resonance

Synthetic polymers are mixtures of different length chains, and their chain length and chain conformation is often experimentally characterized by ensemble averages. We demonstrate that Double-Electron-Electron-Resonance (DEER) spectroscopy can reveal the chain length distribution, and chain conformation and flexibility of the individual n-mers in oligo-(9,9-dioctylfluorene) from controlled Suzuki-Miyaura Coupling Polymerization (cSMCP). The required spin-labeled chain ends were introduced efficiently via a TEMPO-substituted initiator and chain terminating agent, respectively, with an in situ catalyst system. Individual precise chain length oligomers as reference materials were obtained by a stepwise approach. Chain length distribution, chain conformation and flexibility can also be accessed within poly(fluorene) nanoparticles.

In-Cell Double Electron–Electron Resonance at Nanomolar Protein Concentrations

2021 ◽  
pp. 3679-3684
Author(s):  
Svetlana Kucher ◽  
Christina Elsner ◽  
Mariya Safonova ◽  
Stefano Maffini ◽  
Enrica Bordignon
Keyword(s):  
Electron Resonance ◽  
Double Electron ◽  
Double Electron Electron Resonance

Distance Determination in Proteins insideXenopus laevisOocytes by Double Electron−Electron Resonance Experiments

2010 ◽  
Vol 132 (24) ◽  
pp. 8228-8229 ◽  
Author(s):  
Ryuji Igarashi ◽  
Tomomi Sakai ◽  
Hideyuki Hara ◽  
Takeshi Tenno ◽  
Toshiaki Tanaka ◽  
...  
Keyword(s):  
Distance Determination ◽  
Electron Resonance ◽  
Double Electron ◽  
Double Electron Electron Resonance

Tonoplast and plasma membrane ATPases from maize lines of high or low vigour

1992 ◽  
Vol 2 (2) ◽  
pp. 105-111 ◽  
Author(s):  
S. Sánchez-Nieto ◽  
R. Rodríguez-Sotres ◽  
P. González-Romo ◽  
I. Bernal-Lugo ◽  
M. Gavilanes-Ruíz
Keyword(s):  
Zea Mays ◽  
Plasma Membrane ◽  
Acid Phosphatase ◽  
Atpase Activity ◽  
Kinetic Analysis ◽  
Atp Hydrolysis ◽  
Membrane Atpases ◽  
Substrate Concentrations ◽  
Tonoplast Atpase ◽  
Specific Inhibitors

AbstractThe effectiveness of ATPase in germinated seed may play an important role in the vigour of germination. The activities of tonoplast and plasma membrane ATPases in two maize (Zea mays L.) lines with different vigour of germination were determined. ATP hydrolysis was measured in microsomal fractions from coleoptiles along with the responses to specific inhibitors for the plasma membrane, tonoplast and mitochondrial ATPases as well as for acid phosphatase. Nitrate-sensitive ATPase activity was 1.5–3.0 times lower in the low-vigour line than in the high-vigour line. Kinetic analysis of ATP hydrolysis at different substrate concentrations revealed the existence of two enzymes in the microsomal fractions of the two lines. The Vmax of enzyme 1 in the low-vigour line was a third of that in the high-vigour line. This enzyme was identified as the nitrate-sensitive or tonoplast ATPase on the basis of measurements of ATP hydrolysis in the presence of specific inhibitors at high (8.12mm) and low (0.77mm) ATP concentrations.

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