Conformational equilibrium of an ABC transporter analyzed by luminescence resonance energy transferZoghbi, Nouel Barreto, Hernandez
Biophys J (2025)
Abstract: Humans have three known ATP-binding cassette (ABC) transporters in the inner mitochondrial membrane (ABCB7, ABCB8, and ABCB10). ABCB10, the most studied of them thus far, is essential for normal red blood cell development and protection against oxidative stress, and it was recently found to export biliverdin, a heme degradation product with antioxidant properties. The molecular mechanism underlying the function of ABC transporters remains controversial. Their nucleotide binding domains (NBDs) must dimerize to hydrolyze ATP, but capturing the transporters in such conformation for structural studies has been experimentally difficult, especially for ABCB10 and related eukaryotic transporters. Purified transporters are commonly studied in detergent micelles, or after their reconstitution in nanodiscs, usually at nonphysiological temperature and using nonhydrolyzable ATP analogs or mutations that prevent ATP hydrolysis. Here, we have used luminescence resonance energy transfer to evaluate the effect of experimental conditions on the NBD dimerization of ABCB10. Our results indicate that all conditions used for determination of currently available ABCB10 structures have failed to induce NBD dimerization. ABCB10 in detergent responded only to MgATP at 37°C, whereas reconstituted protein shifted toward dimeric NBDs more easily, including in response to MgAMP-PNP and even present NBD dimerization with MgATP at room temperature. The nanodisc's size affects the nucleotide-free conformational equilibrium of ABCB10 and the response to ATP in the absence of magnesium, but for all analyzed sizes (scaffold proteins MSP1D1, MSP1E3D1, and MSP2N2), a conformation with dimeric NBDs is clearly preferred during active ATP hydrolysis (MgATP, 37°C). These results highlight the sensitivity of this human ABC transporter to experimental conditions and the need for a more cautious interpretation of structural models obtained under far from physiological conditions. A dimeric NBD conformation that has been elusive in previous studies seems to be dominant during MgATP hydrolysis at physiological temperature.Copyright © 2025 Biophysical Society. Published by Elsevier Inc. All rights reserved.
ABC transporter activity is affected by the size of lipid nanodiscsNouel Barreto, Cuello, Zoghbi
FEBS Lett (2025) 599 (4), 502-511
Abstract: Lipid nanodiscs have become a widely used approach for studying membrane proteins thanks to several advantages they offer. They have been especially useful for studying ABC transporters, despite the growing concern about the possible restriction of the conformational changes of the transporters due to the small size of the discs. Here, we performed a systematic study to determine the effect of the nanodisc size on the ATPase activity of model ABC transporters from human, plant, and bacteria. Our data confirm that the activity of the transporters and their response to regulatory molecules is affected by the nanodisc size. Our findings suggest the use of larger membrane scaffold proteins (MSPs), such as MSP2N2 nanodiscs, to minimize alterations caused by the commonly used small MSP1D1.© 2025 The Author(s). FEBS Letters published by John Wiley & Sons Ltd on behalf of Federation of European Biochemical Societies.
Electrochemical studies of the mitochondrial ROMK2 potassium channel activity reconstituted into the free-standing and tethered bilayer lipid membranesStefanowska, Koprowski, Bednarczyk
et alBioelectrochemistry (2023) 151, 108372
Abstract: The renal-outer-medullary‑potassium (ROMK2) channel modulates potassium transport in the kidney. It has been postulated that the ROMK2 is the pore-forming subunit of the mitochondrial ATP-sensitive potassium channel as a mediator of cardioprotection. In this study, cell-free synthesis of the ROMK2 was performed in presence of membrane scaffold protein (MSP1D1) nanodiscs. Activity measurements were achieved after channel reconstitution into the planar lipid bilayer and tethered bilayer lipid membranes. Both methods allowed for monitoring of channel function, verified with channel blocking and activation/re-activation experiments. The primary function of the mitochondrial potassium channels is to regulate the potential of the mitochondrial membrane, which allows them to play an important role in cytoprotection. This work focuses on obtaining the ROMK2 using a cell-free expression system, followed by the incorporation of the channel protein into the lipid bilayer and studying the influence of voltage changes and molecular modulators on channel activity. Channel activity was measured after its reconstitution into two models of lipid bilayers - BLM (Bilayer Lipid Membrane) and tBLM (Tethered Bilayer Lipid Membrane) deposited on a solid gold electrode. These two model membranes and electrochemical measurements made it possible to measure the flux of K+ ions in the presence of channel modulators.Copyright © 2023 Elsevier B.V. All rights reserved.
Structures and Dynamics of Anionic Lipoprotein NanodiscsSweeney, Krueger, Sen
et alJ Phys Chem B (2022) 126 (15), 2850-2862
Abstract: Nanolipoprotein particles known as nanodiscs (NDs) have emerged as versatile and powerful tools for the stabilization of membrane proteins permitting a plethora of structural and biophysical studies. Part of their allure is their flexibility to accommodate many types of lipids and precise control of the composition. However, little is known about how variations in lipid composition impact their structures and dynamics. Herein, we investigate how the introduction of the anionic lipid POPG into POPC NDs impacts these features. Small-angle X-ray and neutron scattering (SAXS and SANS) of variable-composition NDs are complemented with molecular dynamics simulations to interrogate how increasing the concern of POPG impacts the ND shape, structure of the lipid core, and the dynamics of the popular membrane scaffold protein, MSP1D1(-). A convenient benefit of including POPG is that it eliminates D2O-induced aggregation observed in pure POPC NDs, permitting studies by SANS at multiple contrasts. SAXS and SANS data could be globally fit to a stacked elliptical cylinder model as well as an extension of the model that accounts for membrane curvature. Fitting to both models supports that the introduction of POPG results in strongly elliptical NDs; however, MD simulations predict the curvature of the membrane, thereby supporting the use of the latter model. Trends in the model-independent parameters suggest that increases in POPG reduce the conformational heterogeneity of the MSP1D1(-), which is in agreement with MD simulations that show that the incorporation of sufficient POPG suppresses disengagement of the N-terminal helix from the lipid core. These studies highlight novel structural changes in NDs in response to an anionic lipid and will inform the interpretation of future structural studies of membrane proteins embedded in NDs of mixed lipid composition.
膜杰作
Star Staining
