Dynamics of SLC25A51 Reveal Preference for Oxidized NAD+ and Substrate Led Transport
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ABSTRACT:
SUBMITTER: Shivansh Goyal
PROVIDER: S-SCDT-10_15252-EMBR_202256596 | biostudies-other |
REPOSITORIES: biostudies-other
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Project description: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.
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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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Project description:SMYD3, a SET and MYND domain containing lysine methyltransferase, catalyzes the transfer of the methyl group from a methyl donor onto the Nε group of a lysine residue in the substrate protein. Methylation of MAP3 kinase kinase (MAP3K2) by SMYD3 has been implicated in Ras-driven tumorigenesis. The crystal structure of SMYD3 in complex with MAP3K2 peptide reveals a shallow hydrophobic pocket (P-2), which accommodates the binding of a phenylalanine residue at the -2 position of the substrate (F258) is a crucial determinant of substrate specificity of SMYD3. To better understand the substrate preference of SMYD3 at the -2 position, molecular dynamics (MD) simulations and the MM/GBSA method were performed on the crystal structure of SMYD3-MAP3K2 complex (PDB: 5EX0) after substitution of F258 residue of MAP3K2 to each of the other 19 natural residues, respectively. Binding free energy calculations reveal that the P-2 pocket prefers an aromatic hydrophobic group and none of the substitutions behave better than the wild-type phenylalanine residue does. Furthermore, we investigated the structure-activity relationships (SAR) of a series of non-natural phenylalanine derivative substitutions at the -2 position and found that quite a few modifications on the sidechain of F258 residue could strengthen its binding to the P-2 pocket of SMYD3. These explorations provide insights into developing novel SMYD3 inhibitors with high potency and high selectivity against MAP3K2 and cancer.
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Project description:We developed and studied a chemical reaction-transport model for the production of zinc oxide (ZnO) with diethylzinc (DEZn) and oxygen (O2). It was confirmed that a large number of ZnO particles were generated during the growth process by testing the internal particles of the cavity by X-ray diffraction. The formation of Zn3O3 in the gas phase reaction was simulated using density functional theory, and the effect of nucleation and formation of nanoparticles on the growth of the films was revealed. We also speculate that the adsorption of Zn-containing gas on the wall is the main route by which a ZnO film is formed. The mechanism calculated by quantum chemistry was applied in computational fluid dynamics (CFD) simulations using Fluent14.0 software, and the concentration distribution and gas reaction path of the reaction chamber were calculated and analyzed. Finally, a 9 gas phase reaction model and an 8 surface reaction model were established. Together with the transport model, a complete chemical reaction-transport reaction model was constructed for the ZnO-MOCVD cavity. The validity of the model was verified, and the optimum temperature range of DEZn and oxygen-stabilized growth of ZnO films was determined to be 673-873 K. Using the results of the chemical reaction transport model, the geometry and operation parameters of the reactor can be optimized to improve the characteristics of the epitaxial layer.
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Project description:Fasting hyperglycemia in diabetes mellitus is caused by unregulated glucagon secretion that activates gluconeogenesis (GNG) and increases the use of pyruvate, lactate, amino acids, and glycerol. Studies of GNG in hepatocytes, however, tend to test a limited number of substrates at nonphysiologic concentrations. Therefore, we treated cultured primary hepatocytes with three identical substrate mixtures of pyruvate/lactate, glutamine, and glycerol at serum fasting concentrations, where a different U-13C- or 2-13C-labeled substrate was substituted in each mix. In the absence of glucagon stimulation, 80% of the glucose produced in primary hepatocytes incorporated either one or two 13C-labeled glycerol molecules in a 1:1 ratio, reflecting the high overall activity of this pathway. In contrast, glucose produced from 13C-labeled pyruvate/lactate or glutamine rarely incorporated two labeled molecules. While glucagon increased the glycerol and pyruvate/lactate contributions to glucose carbon by 1.6- and 1.8-fold, respectively, the glutamine contribution to glucose carbon was increased 6.4-fold in primary hepatocytes. To account for substrate 13C carbon loss during metabolism, we also performed a metabolic flux analysis, which confirmed that the majority of glucose carbon produced by primary hepatocytes was from glycerol. In vivo studies using a PKA-activation mouse model that represents elevated glucagon activity confirmed that most circulating lactate carbons originated from glycerol, but very little glycerol was derived from lactate carbons, reflecting glycerol's importance as a carbon donor to GNG. Given the diverse entry points for GNG substrates, hepatic glucagon action is unlikely to be due to a single mechanism.
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Project description:FocA belongs to the formate-nitrate transporter family and plays an essential role in the export and uptake of formate in organisms. According to the available crystal structures, the N-terminal residues of FocA are structurally featureless at physiological conditions but at reduced pH form helices to harbor the cytoplasmic entrance of the substrate permeation pathway, which apparently explains the cessation of electrical signal observed in electrophysiological experiments. In this work, we found by structural analysis and molecular dynamics simulations that those N-terminal helices cannot effectively preclude the substrate permeation. Equilibrium simulations and thermodynamic calculations suggest that FocA is permeable to both formate and formic acid, the latter of which is transparent to electrophysiological studies as an electrically neutral species. Hence, the cease of electrical current at acidic pH may be caused by the change of the transported substrate from formate to formic acid. In addition, the mechanism of formate export at physiological pH is discussed.
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Project description:The ATPase activity of the maltose transporter (MalFGK(2)) is dependent on interactions with the maltose-binding protein (MBP). To determine whether direct interactions between the translocated sugar and MalFGK(2) are important for the regulation of ATP hydrolysis, we used an MBP mutant (sMBP) that is able to bind either maltose or sucrose. We observed that maltose- and sucrose-bound sMBP stimulate equal levels of MalFGK(2) ATPase activity. Therefore, the ATPase activity of MalFGK(2) is coupled to translocation of maltose solely by interactions between MalFGK(2) and MBP. For both maltose and sucrose, the ability of sMBP to stimulate the MalFGK(2) ATPase was greatly reduced compared with wild-type MBP, indicating that the mutations in sMBP have interfered with important interactions between MBP and MalFGK(2). High resolution crystal structure analysis of sMBP shows that in the closed conformation with bound sucrose, three of four mutations are buried, and the fourth causes only a minor change in the accessible surface. In contrast, in the open form of sMBP, all of the mutations are accessible, and the main chain of Tyr(62)-Gly(69) is destabilized and occupies an alternative conformation due to the W62Y mutation. On this basis, the compromised ability of sMBP to stimulate ATP hydrolysis by MalFGK(2) is most likely due to a disruption of interactions between MalFGK(2) and the open, rather than the closed, conformation of sMBP. Modeling the open sMBP structure bound to MalFGK(2) in the transition state for ATP hydrolysis points to an important site of interaction and suggests a mechanism for coupling ATP hydrolysis to substrate translocation that is independent of the exact structure of the substrate.
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Project description:Understanding the substrate recognition mechanism of ?-secretase is a key step for establishing substrate-specific inhibition of amyloid ?-protein (A?) production. However, it is widely believed that ?-secretase is a promiscuous protease and that its substrate-specific inhibition is elusive. Here we show that ?-secretase distinguishes the ectodomain length of substrates and preferentially captures and cleaves substrates containing a short ectodomain. We also show that a subset of peptides containing the CDCYCxxxxCxCxSC motif binds to the amino terminus of C99 and inhibits A? production in a substrate-specific manner. Interestingly, these peptides suppress ?-secretase-dependent cleavage of APP, but not that of sialyltransferase 1. Most importantly, intraperitoneal administration of peptides into mice results in a significant reduction in cerebral A? levels. This report provides direct evidence of the substrate preference of ?-secretase and its mechanism. Our results demonstrate that the ectodomain of C99 is a potent target for substrate-specific anti-A? therapeutics to combat Alzheimer's disease.
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Project description:Alphaherpesviruses such as herpes simplex virus and pseudorabies virus (PRV) are neuroinvasive double-stranded DNA (dsDNA) viruses that establish lifelong latency in peripheral nervous system (PNS) neurons of their native hosts. Following reactivation, infection can spread back to the initial mucosal site of infection or, in rare cases, to the central nervous system, with usually serious outcomes. During entry and egress, viral capsids depend on microtubule-based molecular motors for efficient and fast transport. In axons of PNS neurons, cytoplasmic dynein provides force for retrograde movements toward the soma, and kinesins move cargo in the opposite, anterograde direction. The dynamic properties of virus particles in cells can be imaged by fluorescent protein fusions to the small capsid protein VP26, which are incorporated into capsids. However, single-color fluorescent protein tags fail to distinguish the virus inoculum from progeny. Therefore, we established a dual-color system by growing a recombinant PRV expressing a red fluorescent VP26 fusion (PRV180) on a stable cell line expressing a green VP26 fusion (PK15-mNG-VP26). The resulting dual-color virus preparation (PRV180G) contains capsids tagged with both red and green fluorescent proteins, and 97% of particles contain detectable levels of mNeonGreen (mNG)-tagged VP26. After replication in neuronal cells, all PRV180G progeny exclusively contain monomeric red fluorescent protein (mRFP)-VP26-tagged capsids. We used PRV180G for an analysis of axonal capsid transport dynamics in PNS neurons. Fast dual-color total internal reflection fluorescence (TIRF) microscopy, single-particle tracking, and motility analyses reveal robust, bidirectional capsid motility mediated by cytoplasmic dynein and kinesin during entry, whereas egressing progeny particles are transported exclusively by kinesins. IMPORTANCE Alphaherpesviruses are neuroinvasive viruses that infect the peripheral nervous system (PNS) of infected hosts as an integral part of their life cycle. Establishment of a quiescent or latent infection in PNS neurons is a hallmark of most alphaherpesviruses. Spread of infection to the central nervous system is surprisingly rare in natural hosts but can be fatal. Pseudorabies virus (PRV) is a broad-host-range swine alphaherpesvirus that enters neuronal cells and utilizes intracellular transport processes to establish infection and to spread between cells. By using a virus preparation with fluorescent viral capsids that change color depending on the stage of the infectious cycle, we find that during entry, axons of PNS neurons support robust, bidirectional capsid motility, similar to cellular cargo, toward the cell body. In contrast, progeny particles appear to be transported unidirectionally by kinesin motors toward distal egress sites.
| S-EPMC5068546 | biostudies-literature
Project description:The nicotinamide adenine dinucleotide (NAD+/NADH) pair is a cofactor in redox reactions and is particularly critical in mitochondria as it connects substrate oxidation by the tricarboxylic acid (TCA) cycle to adenosine triphosphate generation by the electron transport chain (ETC) and oxidative phosphorylation. While a mitochondrial NAD+ transporter has been identified in yeast, how NAD enters mitochondria in metazoans is unknown. Here, we mine gene essentiality data from human cell lines to identify MCART1 (SLC25A51) as coessential with ETC components. MCART1-null cells have large decreases in TCA cycle flux, mitochondrial respiration, ETC complex I activity, and mitochondrial levels of NAD+ and NADH. Isolated mitochondria from cells lacking or overexpressing MCART1 have greatly decreased or increased NAD uptake in vitro, respectively. Moreover, MCART1 and NDT1, a yeast mitochondrial NAD+ transporter, can functionally complement for each other. Thus, we propose that MCART1 is the long sought mitochondrial transporter for NAD in human cells.
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