| 1. |
- Choudhury, Koushik, et al.
(författare)
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An open state of a voltage-gated sodium channel involving a p-helix and conserved pore-facing asparagine
- 2022
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Ingår i: Biophysical Journal. - : Elsevier BV. - 0006-3495 .- 1542-0086. ; 121:1, s. 11-22
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Tidskriftsartikel (refereegranskat)abstract
- Voltage-gated sodium (Nav) channels play critical roles in propagating action potentials and otherwise manipulating ionic gradients in excitable cells. These channels open in response to membrane depolarization, selectively permeating sodium ions until rapidly inactivating. Structural characterization of the gating cycle in this channel family has proved challenging, particularly due to the transient nature of the open state. A structure from the bacterium Magnetococcus marinus Nav (NavMs) was initially proposed to be open, based on its pore diameter and voltage-sensor conformation. However, the functional annotation of this model, and the structural details of the open state, remain disputed. In this work, we used molecular modeling and simulations to test possible open-state models of NavMs. The full-length experimental structure, termed here the cc-model, was consistently dehydrated at the activation gate, indicating an inability to conduct ions. Based on a spontaneous transition observed in extended simulations, and sequence/structure comparison to other Nav channels, we built an alternative p-model featuring a helix transition and the rotation of a conserved asparagine residue into the activation gate. Pore hydration, ion permeation, and state-dependent drug binding in this model were consistent with an open functional state. This work thus offers both a functional annotation of the full-length NavMs structure and a detailed model for a stable Nav open state, with potential conservation in diverse ion-channel families.
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| 2. |
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| 3. |
- McComas, Sarah, et al.
(författare)
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Determinants of sugar-induced influx in the mammalian fructose transporter GLUT5
- 2023
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Ingår i: eLIFE. - : eLife Sciences Publications, Ltd. - 2050-084X. ; 12
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Tidskriftsartikel (refereegranskat)abstract
- In mammals, glucose transporters (GLUT) control organism-wide blood-glucose homeostasis. In human, this is accomplished by 14 different GLUT isoforms, that transport glucose and other monosaccharides with varying substrate preferences and kinetics. Nevertheless, there is little difference between the sugar-coordinating residues in the GLUT proteins and even the malarial Plasmodium falciparum transporter PfHT1, which is uniquely able to transport a wide range of different sugars. PfHT1 was captured in an intermediate 'occluded' state, revealing how the extracellular gating helix TM7b has moved to break and occlude the sugar-binding site. Sequence difference and kinetics indicated that the TM7b gating helix dynamics and interactions likely evolved to enable substrate promiscuity in PfHT1, rather than the sugar-binding site itself. It was unclear, however, if the TM7b structural transitions observed in PfHT1 would be similar in the other GLUT proteins. Here, using enhanced sampling molecular dynamics simulations, we show that the fructose transporter GLUT5 spontaneously transitions through an occluded state that closely resembles PfHT1. The coordination of D-fructose lowers the energetic barriers between the outward- and inward-facing states, and the observed binding mode for D-fructose is consistent with biochemical analysis. Rather than a substrate-binding site that achieves strict specificity by having a high affinity for the substrate, we conclude GLUT proteins have allosterically coupled sugar binding with an extracellular gate that forms the high-affinity transition-state instead. This substrate-coupling pathway presumably enables the catalysis of fast sugar flux at physiological relevant blood-glucose concentrations.
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| 4. |
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| 5. |
- McComas, Sarah, 1992-
(författare)
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The molecular basis for substrate recognition and gating in sugar transporters
- 2023
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Sugar is a vital sustenance for most forms of life. For a cell to take up sugar, specialized transport proteins embedded into the membrane bilayer known as sugar porters, are required. Dysfunction of sugar porters is associated with some metabolic diseases, and their expression is upregulated in many cancers as they typically require more sugar than normal cells. Furthermore, sugar porters also play a role in the vitality of the malaria parasite.The mechanism of sugar transport is known as a rocker-switch alternating access mechanism. Simplistically, sugar binds between two similar domains on the outside of a sugar transporter and the domains then move around the sugar, so the sugar is exposed to the inside. During this domain movement, protein mass will block the sugar binding site from both outside and inside, forming the occluded state which is essential to ensure no substrate leakage during transport. Despite this relatively simple model of transport, little is known about how different sugar porters display diverse substrate specificity, affinity, and turnover.In the four papers making up this thesis, we structurally characterize missing pieces of the sugar transport cycle, identify how these states are connected with simulations, and assess factors contributing to sugar transport by functional assays. With simulations, we show how sugar catalyzes conformational change by interacting with the occluded state. We demonstrate our functional proteoliposome-based transport assay, which allows us to measure the effect of protein mutations, inhibitors, and lipid influences in sugar recognition and turnover. Characterization of the malaria parasite hexose transporter PfHT1 has allowed us to understand antimalarial inhibitor specificity against this protein which could have implications in combating the disease, as well as pharmacological control of sugar porters in general.
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| 6. |
- Mitrovic, Darko, et al.
(författare)
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Reconstructing the transport cycle in the sugar porter superfamily using coevolution-powered machine learning
- 2023
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Ingår i: eLIFE. - : eLife Sciences Publications, Ltd. - 2050-084X. ; 12
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Tidskriftsartikel (refereegranskat)abstract
- Sugar porters (SPs) represent the largest group of secondary-active transporters. Some members, such as the glucose transporters (GLUTs), are well known for their role in maintaining blood glucose homeostasis in mammals, with their expression upregulated in many types of cancers. Because only a few sugar porter structures have been determined, mechanistic models have been constructed by piecing together structural states of distantly related proteins. Current GLUT transport models are predominantly descriptive and oversimplified. Here, we have combined coevolution analysis and comparative modeling, to predict structures of the entire sugar porter superfamily in each state of the transport cycle. We have analyzed the state-specific contacts inferred from coevolving residue pairs and shown how this information can be used to rapidly generate free-energy landscapes consistent with experimental estimates, as illustrated here for the mammalian fructose transporter GLUT5. By comparing many different sugar porter models and scrutinizing their sequence, we have been able to define the molecular determinants of the transport cycle, which are conserved throughout the sugar porter superfamily. We have also been able to highlight differences leading to the emergence of proton-coupling, validating, and extending the previously proposed latch mechanism. Our computational approach is transferable to any transporter, and to other protein families in general.
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| 7. |
- Mühleip, Alexander, et al.
(författare)
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Structure of a mitochondrial ATP synthase with bound native cardiolipin
- 2019
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Ingår i: eLIFE. - 2050-084X. ; 8
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Tidskriftsartikel (refereegranskat)abstract
- The mitochondrial ATP synthase fuels eukaryotic cells with chemical energy. Here we report the cryo-EM structure of a divergent ATP synthase dimer from mitochondria of Euglena gracilis, a member of the phylum Euglenozoa that also includes human parasites. It features 29 different subunits, 8 of which are newly identified. The membrane region was determined to 2.8 angstrom resolution, enabling the identification of 37 associated lipids, including 25 cardiolipins, which provides insight into protein-lipid interactions and their functional roles. The rotor-stator interface comprises four membrane-embedded horizontal helices, including a distinct subunit a. The dimer interface is formed entirely by phylum-specific components, and a peripherally associated subcomplex contributes to the membrane curvature. The central and peripheral stalks directly interact with each other. Last, the ATPase inhibitory factor 1 (IF1) binds in a mode that is different from human, but conserved in Trypanosomatids.
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| 10. |
- Qureshi, Abdul Aziz, et al.
(författare)
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The molecular basis for sugar import in malaria parasites
- 2020
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Ingår i: Nature. - : Springer Science and Business Media LLC. - 0028-0836 .- 1476-4687. ; 578:7794, s. 321-325
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Tidskriftsartikel (refereegranskat)abstract
- Elucidating the mechanism of sugar import requires a molecular understanding of how transporters couple sugar binding and gating events. Whereas mammalian glucose transporters (GLUTs) are specialists1, the hexose transporter from the malaria parasite Plasmodium falciparum PfHT12,3 has acquired the ability to transport both glucose and fructose sugars as efficiently as the dedicated glucose (GLUT3) and fructose (GLUT5) transporters. Here, to establish the molecular basis of sugar promiscuity in malaria parasites, we determined the crystal structure of PfHT1 in complex with d-glucose at a resolution of 3.6 Å. We found that the sugar-binding site in PfHT1 is very similar to those of the distantly related GLUT3 and GLUT5 structures4,5. Nevertheless, engineered PfHT1 mutations made to match GLUT sugar-binding sites did not shift sugar preferences. The extracellular substrate-gating helix TM7b in PfHT1 was positioned in a fully occluded conformation, providing a unique glimpse into how sugar binding and gating are coupled. We determined that polar contacts between TM7b and TM1 (located about 15 Å from d-glucose) are just as critical for transport as the residues that directly coordinate d-glucose, which demonstrates a strong allosteric coupling between sugar binding and gating. We conclude that PfHT1 has achieved substrate promiscuity not by modifying its sugar-binding site, but instead by evolving substrate-gating dynamics.
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| 11. |
- Suades, Albert, et al.
(författare)
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Establishing mammalian GLUT kinetics and lipid composition influences in a reconstituted-liposome system
- 2023
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Ingår i: Nature Communications. - : Springer Nature. - 2041-1723. ; 14:1
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Tidskriftsartikel (refereegranskat)abstract
- Transport assays using purified glucose transporters (GLUTs) have proven to be difficult to implement, hampering deeper mechanistic insights. Here the authors have optimized a transport assay in liposomes that will provide insight to study other membrane transport proteins. Glucose transporters (GLUTs) are essential for organism-wide glucose homeostasis in mammals, and their dysfunction is associated with numerous diseases, such as diabetes and cancer. Despite structural advances, transport assays using purified GLUTs have proven to be difficult to implement, hampering deeper mechanistic insights. Here, we have optimized a transport assay in liposomes for the fructose-specific isoform GLUT5. By combining lipidomic analysis with native MS and thermal-shift assays, we replicate the GLUT5 transport activities seen in crude lipids using a small number of synthetic lipids. We conclude that GLUT5 is only active under a specific range of membrane fluidity, and that human GLUT1-4 prefers a similar lipid composition to GLUT5. Although GLUT3 is designated as the high-affinity glucose transporter, in vitro D-glucose kinetics demonstrates that GLUT1 and GLUT3 actually have a similar K-M,K- but GLUT3 has a higher turnover. Interestingly, GLUT4 has a high K-M for D-glucose and yet a very slow turnover, which may have evolved to ensure uptake regulation by insulin-dependent trafficking. Overall, we outline a much-needed transport assay for measuring GLUT kinetics and our analysis implies that high-levels of free fatty acid in membranes, as found in those suffering from metabolic disorders, could directly impair glucose uptake.
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| 12. |
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