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Dynamics of SLC25A51 reveal preference for oxidized NAD⁺ and substrate led transport.

ABSTRACT: SLC25A51 is a member of the mitochondrial carrier family (MCF) but lacks key residues that contribute to the mechanism of other nucleotide MCF transporters. Thus, how SLC25A51 transports NAD⁺ across the inner mitochondrial membrane remains unclear. To elucidate its mechanism, we use Molecular Dynamics simulations to reconstitute SLC25A51 homology models into lipid bilayers and to generate hypotheses to test. We observe spontaneous binding of cardiolipin phospholipids to three distinct sites on the exterior of SLC25A51's central pore and find that mutation of these sites impairs cardiolipin binding and transporter activity. We also observe that stable formation of the required matrix gate is controlled by a single salt bridge. We identify binding sites in SLC25A51 for NAD⁺ and show that its selectivity for NAD⁺ is guided by an electrostatic interaction between the charged nicotinamide ring in the ligand and a negatively charged patch in the pore. In turn, interaction of NAD⁺ with interior residue E132 guides the ligand to dynamically engage and weaken the salt bridge gate, representing a ligand-induced initiation of transport.

SUBMITTER: Goyal S

PROVIDER: S-EPMC10561365 | biostudies-literature | 2023 Oct

REPOSITORIES: biostudies-literature

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Dynamics of SLC25A51 reveal preference for oxidized NAD<sup>+</sup> and substrate led transport.

Goyal Shivansh S Paspureddi Akhilesh A Lu Mu-Jie MJ Chan Hsin-Ru HR Lyons Scott N SN Wilson Crystal N CN Niere Marc M Ziegler Mathias M Cambronne Xiaolu A XA

EMBO reports 20230814 10

SLC25A51 is a member of the mitochondrial carrier family (MCF) but lacks key residues that contribute to the mechanism of other nucleotide MCF transporters. Thus, how SLC25A51 transports NAD<sup>+</sup> across the inner mitochondrial membrane remains unclear. To elucidate its mechanism, we use Molecular Dynamics simulations to reconstitute SLC25A51 homology models into lipid bilayers and to generate hypotheses to test. We observe spontaneous binding of cardiolipin phospholipids to three distinct ...[more]

PMID: 37575034

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Project description:IntroductionNicotinamide adenine dinucleotide (NAD) is a coenzyme central to metabolism and energy production. NAD+-dependent deacetylase sirtuin 3 (SIRT3) regulates the acetylation levels of mitochondrial proteins that are involved in mitochondrial homeostasis. Fasting up-regulates hepatic SIRT3 activity, which requires mitochondrial NAD+. What is the mechanism, then, to transport more NAD+ into mitochondria to sustain enhanced SIRT3 activity during fasting?ObjectiveSLC25A51 is a recently discovered mitochondrial NAD+ transporter. We tested the hypothesis that, during fasting, increased expression of SLC25A51 is needed for enhanced mitochondrial NAD+ uptake to sustain SIRT3 activity. Because the fasting-fed cycle and circadian rhythm are closely linked, we further tested the hypothesis that SLC25A51 is a circadian regulated gene.MethodsWe examined Slc25a51 expression in the liver of fasted mice, and examined its circadian rhythm in wild-type mice and those with liver-specific deletion of the clock gene BMAL1 (LKO). We suppressed Slc25a51 expression in hepatocytes and the mouse liver using shRNA-mediated knockdown, and then examined mitochondrial NAD+ levels, SIRT3 activities, and acetylation levels of SIRT3 target proteins (IDH2 and ACADL). We measured mitochondrial oxygen consumption rate using Seahorse analysis in hepatocytes with reduced Slc25a51 expression.ResultsWe found that fasting induced the hepatic expression of Slc25a51, and its expression showed a circadian rhythm-like pattern that was disrupted in LKO mice. Reduced expression of Slc25a51 in hepatocytes decreased mitochondrial NAD+ levels and SIRT3 activity, reflected by increased acetylation of SIRT3 targets. Slc25a51 knockdown reduced the oxygen consumption rate in intact hepatocytes. Mice with reduced Slc25a51 expression in the liver manifested reduced hepatic mitochondrial NAD+ levels, hepatic steatosis and hypertriglyceridemia.ConclusionsSlc25a51 is a fasting-induced gene that is needed for hepatic SIRT3 functions.

Project description:BackgroundGlucose transporter 4 (GLUT4) is an insulin facilitated glucose transporter that plays an important role in maintaining blood glucose homeostasis. GLUT4 is sequestered into intracellular vesicles in unstimulated cells and translocated to the plasma membrane by various stimuli. Understanding the structural details of GLUT4 will provide insights into the mechanism of glucose transport and its regulation. To date, a crystal structure for GLUT4 is not available. However, earlier work from our laboratory proposed a well validated homology model for GLUT4 based on the experimental data available on GLUT1 and the crystal structure data obtained from the glycerol 3-phosphate transporter.Methodology/principal findingsIn the present study, the dynamic behavior of GLUT4 in a membrane environment was analyzed using three forms of GLUT4 (apo, substrate and ATP-substrate bound states). Apo form simulation analysis revealed an extracellular open conformation of GLUT4 in the membrane favoring easy exofacial binding of substrate. Simulation studies with the substrate bound form proposed a stable state of GLUT4 with glucose, which can be a substrate-occluded state of the transporter. Principal component analysis suggested a clockwise movement for the domains in the apo form, whereas ATP substrate-bound form induced an anti-clockwise rotation. Simulation studies suggested distinct conformational changes for the GLUT4 domains in the ATP substrate-bound form and favor a constricted behavior for the transport channel. Various inter-domain hydrogen bonds and switching of a salt-bridge network from E345-R350-E409 to E345-R169-E409 contributed to this ATP-mediated channel constriction favoring substrate occlusion and prevention of its release into cytoplasm. These data are consistent with the biochemical studies, suggesting an inhibitory role for ATP in GLUT-mediated glucose transport.Conclusions/significanceIn the absence of a crystal structure for any glucose transporter, this study provides mechanistic details of the conformational changes in GLUT4 induced by substrate and its regulator.

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Dynamics of SLC25A51 reveal preference for oxidized NAD⁺ and substrate led transport.

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Dynamics of SLC25A51 reveal preference for oxidized NAD<sup>+</sup> and substrate led transport.

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Dynamics of SLC25A51 reveal preference for oxidized NAD+ and substrate led transport.

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Dynamics of SLC25A51 reveal preference for oxidized NAD<sup>+</sup> and substrate led transport.

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OmicsDI is part of the ELIXIR infrastructure

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Dynamics of SLC25A51 reveal preference for oxidized NAD⁺ and substrate led transport.