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Publications (10 of 44) Show all publications
Drew, D. & Boudker, O. (2024). Ion and lipid orchestration of secondary active transport. Nature, 626(8001), 963-974
Open this publication in new window or tab >>Ion and lipid orchestration of secondary active transport
2024 (English)In: Nature, ISSN 0028-0836, E-ISSN 1476-4687, Vol. 626, no 8001, p. 963-974Article, review/survey (Refereed) Published
Abstract [en]

Transporting small molecules across cell membranes is an essential process in cell physiology. Many structurally diverse, secondary active transporters harness transmembrane electrochemical gradients of ions to power the uptake or efflux of nutrients, signalling molecules, drugs and other ions across cell membranes. Transporters reside in lipid bilayers on the interface between two aqueous compartments, where they are energized and regulated by symported, antiported and allosteric ions on both sides of the membrane and the membrane bilayer itself. Here we outline the mechanisms by which transporters couple ion and solute fluxes and discuss how structural and mechanistic variations enable them to meet specific physiological needs and adapt to environmental conditions. We then consider how general bilayer properties and specific lipid binding modulate transporter activity. Together, ion gradients and lipid properties ensure the effective transport, regulation and distribution of small molecules across cell membranes.

National Category
Cell Biology
Identifiers
urn:nbn:se:su:diva-227692 (URN)10.1038/s41586-024-07062-3 (DOI)001183983000003 ()38418916 (PubMedID)2-s2.0-85186231471 (Scopus ID)
Available from: 2024-04-05 Created: 2024-04-05 Last updated: 2024-04-05Bibliographically approved
Currie, M. J., Davies, J. S., Scalise, M., Gulati, A., Wright, J. D., Newton-Vesty, M. C., . . . North, R. A. (2024). Structural and biophysical analysis of a Haemophilus influenzae tripartite ATP-independent periplasmic (TRAP) transporter. eLIFE, 12, Article ID RP92307.
Open this publication in new window or tab >>Structural and biophysical analysis of a Haemophilus influenzae tripartite ATP-independent periplasmic (TRAP) transporter
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2024 (English)In: eLIFE, E-ISSN 2050-084X, Vol. 12, article id RP92307Article in journal (Refereed) Published
Abstract [en]

Tripartite ATP-independent periplasmic (TRAP) transporters are secondary-active transporters that receive their substrates via a soluble-binding protein to move bioorganic acids across bacterial or archaeal cell membranes. Recent cryo-electron microscopy (cryo-EM) structures of TRAP transporters provide a broad framework to understand how they work, but the mechanistic details of transport are not yet defined. Here we report the cryo-EM structure of the Haemophilus influenzae N-acetylneuraminate TRAP transporter (HiSiaQM) at 2.99 Å resolution (extending to 2.2 Å at the core), revealing new features. The improved resolution (the previous HiSiaQM structure is 4.7 Å resolution) permits accurate assignment of two Na+ sites and the architecture of the substrate-binding site, consistent with mutagenic and functional data. Moreover, rather than a monomer, the HiSiaQM structure is a homodimer. We observe lipids at the dimer interface, as well as a lipid trapped within the fusion that links the SiaQ and SiaM subunits. We show that the affinity (KD) for the complex between the soluble HiSiaP protein and HiSiaQM is in the micromolar range and that a related SiaP can bind HiSiaQM. This work provides key data that enhances our understanding of the ‘elevator-with-an-operator’ mechanism of TRAP transporters.

Keywords
sialic acid, Neu5Ac, protein-protein interaction, membrane transport proteins, transport mechanism, Other
National Category
Biochemistry and Molecular Biology
Identifiers
urn:nbn:se:su:diva-227954 (URN)10.7554/eLife.92307 (DOI)001162186400001 ()38349818 (PubMedID)2-s2.0-85182017089 (Scopus ID)
Available from: 2024-04-09 Created: 2024-04-09 Last updated: 2024-04-09Bibliographically approved
Mazza, T., Roumeliotis, T. I., Garitta, E., Drew, D., Rashid, S. T., Indiveri, C., . . . Beis, K. (2024). Structural basis for the modulation of MRP2 activity by phosphorylation and drugs. Nature Communications, 15(1), Article ID 1983.
Open this publication in new window or tab >>Structural basis for the modulation of MRP2 activity by phosphorylation and drugs
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2024 (English)In: Nature Communications, E-ISSN 2041-1723, Vol. 15, no 1, article id 1983Article in journal (Refereed) Published
Abstract [en]

Multidrug resistance-associated protein 2 (MRP2/ABCC2) is a polyspecific efflux transporter of organic anions expressed in hepatocyte canalicular membranes. MRP2 dysfunction, in Dubin-Johnson syndrome or by off-target inhibition, for example by the uricosuric drug probenecid, elevates circulating bilirubin glucuronide and is a cause of jaundice. Here, we determine the cryo-EM structure of rat Mrp2 (rMrp2) in an autoinhibited state and in complex with probenecid. The autoinhibited state exhibits an unusual conformation for this class of transporter in which the regulatory domain is folded within the transmembrane domain cavity. In vitro phosphorylation, mass spectrometry and transport assays show that phosphorylation of the regulatory domain relieves this autoinhibition and enhances rMrp2 transport activity. The in vitro data is confirmed in human hepatocyte-like cells, in which inhibition of endogenous kinases also reduces human MRP2 transport activity. The drug-bound state reveals two probenecid binding sites that suggest a dynamic interplay with autoinhibition. Mapping of the Dubin-Johnson mutations onto the rodent structure indicates that many may interfere with the transition between conformational states. The ABC transporter MRP2/ABCC2 is a polyspecific efflux transporter of organic anions expressed in hepatocyte canalicular membranes. Dysfunction leads to Dubin-Johnson syndrome. Here the authors provide structural and biochemical evidence on the modulation of MRP2 by intracellular kinases and inhibition by therapeutic drugs.

National Category
Biochemistry and Molecular Biology
Identifiers
urn:nbn:se:su:diva-228264 (URN)10.1038/s41467-024-46392-8 (DOI)001179853600029 ()38438394 (PubMedID)
Available from: 2024-04-11 Created: 2024-04-11 Last updated: 2024-04-11Bibliographically approved
Asami, J., Park, J.-H., Nomura, Y., Kobayashi, C., Mifune, J., Ishimoto, N., . . . Ohto, U. (2024). Structural basis of hepatitis B virus receptor binding. Nature Structural & Molecular Biology, 31, 447-454
Open this publication in new window or tab >>Structural basis of hepatitis B virus receptor binding
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2024 (English)In: Nature Structural & Molecular Biology, ISSN 1545-9993, E-ISSN 1545-9985, Vol. 31, p. 447-454Article in journal (Refereed) Published
Abstract [en]

Hepatitis B virus (HBV), a leading cause of developing hepatocellular carcinoma affecting more than 290 million people worldwide, is an enveloped DNA virus specifically infecting hepatocytes. Myristoylated preS1 domain of the HBV large surface protein binds to the host receptor sodium-taurocholate cotransporting polypeptide (NTCP), a hepatocellular bile acid transporter, to initiate viral entry. Here, we report the cryogenic-electron microscopy structure of the myristoylated preS1 (residues 2–48) peptide bound to human NTCP. The unexpectedly folded N-terminal half of the peptide embeds deeply into the outward-facing tunnel of NTCP, whereas the C-terminal half formed extensive contacts on the extracellular surface. Our findings reveal an unprecedented induced-fit mechanism for establishing high-affinity virus–host attachment and provide a blueprint for the rational design of anti-HBV drugs targeting virus entry. 

National Category
Biochemistry and Molecular Biology
Identifiers
urn:nbn:se:su:diva-226141 (URN)10.1038/s41594-023-01191-5 (DOI)001143881600001 ()38233573 (PubMedID)2-s2.0-85182420581 (Scopus ID)
Available from: 2024-02-01 Created: 2024-02-01 Last updated: 2024-04-29Bibliographically approved
McComas, S., Reichenbach, T., Mitrovic, D., Alleva, C., Bonaccorsi, M., Delemotte, L., . . . Stockbridge, R. B. (2023). Determinants of sugar-induced influx in the mammalian fructose transporter GLUT5. eLIFE, 12, Article ID e84808.
Open this publication in new window or tab >>Determinants of sugar-induced influx in the mammalian fructose transporter GLUT5
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2023 (English)In: eLIFE, E-ISSN 2050-084X, Vol. 12, article id e84808Article in journal (Refereed) Published
Abstract [en]

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.

National Category
Other Biological Topics
Identifiers
urn:nbn:se:su:diva-220841 (URN)10.7554/eLife.84808 (DOI)001024510300001 ()37405832 (PubMedID)2-s2.0-85163948061 (Scopus ID)
Available from: 2023-09-14 Created: 2023-09-14 Last updated: 2023-11-06Bibliographically approved
Suades, A., Qureshi, A. A., McComas, S., Coincon, M., Rudling, A., Chatzikyriakidou, Y., . . . Drew, D. (2023). Establishing mammalian GLUT kinetics and lipid composition influences in a reconstituted-liposome system. Nature Communications, 14(1)
Open this publication in new window or tab >>Establishing mammalian GLUT kinetics and lipid composition influences in a reconstituted-liposome system
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2023 (English)In: Nature Communications, E-ISSN 2041-1723, Vol. 14, no 1Article in journal (Refereed) Published
Abstract [en]

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.

National Category
Other Natural Sciences
Identifiers
urn:nbn:se:su:diva-221385 (URN)10.1038/s41467-023-39711-y (DOI)001027089000013 ()37429918 (PubMedID)2-s2.0-85164297820 (Scopus ID)
Available from: 2023-09-20 Created: 2023-09-20 Last updated: 2023-10-09Bibliographically approved
Mitrovic, D., McComas, S. E., Alleva, C., Bonaccorsi, M., Drew, D. & Delemotte, L. (2023). Reconstructing the transport cycle in the sugar porter superfamily using coevolution-powered machine learning. eLIFE, 12, Article ID e84805.
Open this publication in new window or tab >>Reconstructing the transport cycle in the sugar porter superfamily using coevolution-powered machine learning
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2023 (English)In: eLIFE, E-ISSN 2050-084X, Vol. 12, article id e84805Article in journal (Refereed) Published
Abstract [en]

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.

National Category
Biophysics
Identifiers
urn:nbn:se:su:diva-223414 (URN)10.7554/eLife.84805 (DOI)001071912700001 ()37405846 (PubMedID)2-s2.0-85164005539 (Scopus ID)
Available from: 2023-11-02 Created: 2023-11-02 Last updated: 2023-11-06Bibliographically approved
Yeo, H., Mehta, V., Gulati, A. & Drew, D. (2023). Structure and electromechanical coupling of a voltage-gated Na+/H+ exchanger. Nature, 623(7985), 193-201
Open this publication in new window or tab >>Structure and electromechanical coupling of a voltage-gated Na+/H+ exchanger
2023 (English)In: Nature, ISSN 0028-0836, E-ISSN 1476-4687, Vol. 623, no 7985, p. 193-201Article in journal (Refereed) Published
Abstract [en]

Voltage-sensing domains control the activation of voltage-gated ion channels, with a few exceptions. One such exception is the sperm-specific Na+/H+ exchanger SLC9C1, which is the only known transporter to be regulated by voltage-sensing domains. After hyperpolarization of sperm flagella, SLC9C1 becomes active, causing pH alkalinization and CatSper Ca2+ channel activation, which drives chemotaxis. SLC9C1 activation is further regulated by cAMP, which is produced by soluble adenyl cyclase (sAC). SLC9C1 is therefore an essential component of the pH–sAC–cAMP signalling pathway in metazoa, required for sperm motility and fertilization. Despite its importance, the molecular basis of SLC9C1 voltage activation is unclear. Here we report cryo-electron microscopy (cryo-EM) structures of sea urchin SLC9C1 in detergent and nanodiscs. We show that the voltage-sensing domains are positioned in an unusual configuration, sandwiching each side of the SLC9C1 homodimer. The S4 segment is very long, 90 Å in length, and connects the voltage-sensing domains to the cytoplasmic cyclic-nucleotide-binding domains. The S4 segment is in the up configuration—the inactive state of SLC9C1. Consistently, although a negatively charged cavity is accessible for Na+ to bind to the ion-transporting domains of SLC9C1, an intracellular helix connected to S4 restricts their movement. On the basis of the differences in the cryo-EM structure of SLC9C1 in the presence of cAMP, we propose that, upon hyperpolarization, the S4 segment moves down, removing this constriction and enabling Na+/H+ exchange.

National Category
Biochemistry and Molecular Biology Cell Biology
Identifiers
urn:nbn:se:su:diva-227689 (URN)10.1038/s41586-023-06518-2 (DOI)001168920600019 ()37880360 (PubMedID)2-s2.0-85174843904 (Scopus ID)
Available from: 2024-04-05 Created: 2024-04-05 Last updated: 2024-04-05Bibliographically approved
Davies, J. S., Currie, M. J., North, R. A., Scalise, M., Wright, J. D., Copping, J. M., . . . Dobson, R. C. J. (2023). Structure and mechanism of a tripartite ATP-independent periplasmic TRAP transporter. Nature Communications, 14(1), Article ID 1120.
Open this publication in new window or tab >>Structure and mechanism of a tripartite ATP-independent periplasmic TRAP transporter
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2023 (English)In: Nature Communications, E-ISSN 2041-1723, Vol. 14, no 1, article id 1120Article in journal (Refereed) Published
Abstract [en]

In bacteria and archaea, tripartite ATP-independent periplasmic (TRAP) transporters uptake essential nutrients. TRAP transporters receive their substrates via a secreted soluble substrate-binding protein. How a sodium ion-driven secondary active transporter is strictly coupled to a substrate-binding protein is poorly understood. Here we report the cryo-EM structure of the sialic acid TRAP transporter SiaQM from Photobacterium profundum at 2.97 angstrom resolution. SiaM comprises a transport domain and a scaffold domain, with the transport domain consisting of helical hairpins as seen in the sodium ion-coupled elevator transporter VcINDY. The SiaQ protein forms intimate contacts with SiaM to extend the size of the scaffold domain, suggesting that TRAP transporters may operate as monomers, rather than the typically observed oligomers for elevator-type transporters. We identify the Na+ and sialic acid binding sites in SiaM and demonstrate a strict dependence on the substrate-binding protein SiaP for uptake. We report the SiaP crystal structure that, together with docking studies, suggest the molecular basis for how sialic acid is delivered to the SiaQM transporter complex. We thus propose a model for substrate transport by TRAP proteins, which we describe herein as an 'elevator-with-an-operator' mechanism. Bacteria and archaea use tripartite ATP-independent periplasmic (TRAP) transporters to import essential nutrients. Davies et al. report a high resolution structure of a TRAP and show that it uses an 'elevator-with-an operator' mechanism.

National Category
Biochemistry and Molecular Biology
Identifiers
urn:nbn:se:su:diva-215855 (URN)10.1038/s41467-023-36590-1 (DOI)000942107800002 ()36849793 (PubMedID)2-s2.0-85148970924 (Scopus ID)
Available from: 2023-03-28 Created: 2023-03-28 Last updated: 2023-03-28Bibliographically approved
Winkelmann, I., Uzdavinys, P., Kenney, I. M., Brock, J., Meier, P. F., Wagner, L.-M., . . . Drew, D. (2022). Crystal structure of the Na+/H+ antiporter NhaA at active pH reveals the mechanistic basis for pH sensing. Nature Communications, 13(1), Article ID 6383.
Open this publication in new window or tab >>Crystal structure of the Na+/H+ antiporter NhaA at active pH reveals the mechanistic basis for pH sensing
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2022 (English)In: Nature Communications, E-ISSN 2041-1723, Vol. 13, no 1, article id 6383Article in journal (Refereed) Published
Abstract [en]

The strict exchange of protons for sodium ions across cell membranes by Na+/H+ exchangers is a fundamental mechanism for cell homeostasis. At active pH, Na+/H+ exchange can be modelled as competition between H+ and Na+ to an ion-binding site, harbouring either one or two aspartic-acid residues. Nevertheless, extensive analysis on the model Na+/H+ antiporter NhaA from Escherichia coli, has shown that residues on the cytoplasmic surface, termed the pH sensor, shifts the pH at which NhaA becomes active. It was unclear how to incorporate the pH senor model into an alternating-access mechanism based on the NhaA structure at inactive pH 4. Here, we report the crystal structure of NhaA at active pH 6.5, and to an improved resolution of 2.2 angstrom. We show that at pH 6.5, residues in the pH sensor rearrange to form new salt-bridge interactions involving key histidine residues that widen the inward-facing cavity. What we now refer to as a pH gate, triggers a conformational change that enables water and Na+ to access the ion-binding site, as supported by molecular dynamics (MD) simulations. Our work highlights a unique, channel-like switch prior to substrate translocation in a secondary-active transporter. 

National Category
Chemical Sciences
Identifiers
urn:nbn:se:su:diva-211628 (URN)10.1038/s41467-022-34120-z (DOI)000874935700009 ()36289233 (PubMedID)2-s2.0-85140814804 (Scopus ID)
Available from: 2022-11-25 Created: 2022-11-25 Last updated: 2023-03-28Bibliographically approved
Organisations
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ORCID iD: ORCID iD iconorcid.org/0000-0001-8866-6349

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