| 1. |
- Currie, Michael J., et al.
(författare)
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Structural and biophysical analysis of a Haemophilus influenzae tripartite ATP-independent periplasmic (TRAP) transporter
- 2024
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Ingår i: ELIFE. - 2050-084X. ; 12
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Tidskriftsartikel (refereegranskat)abstract
- 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 & Aring; resolution (extending to 2.2 & Aring; at the core), revealing new features. The improved resolution (the previous HiSiaQM structure is 4.7 & Aring; 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 (K-D) 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.
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| 2. |
- Manning, Sinclaire M., et al.
(författare)
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Characterization of Two 2 mm detected Optically Obscured Dusty Star-forming Galaxies
- 2022
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Ingår i: Astrophysical Journal. - : American Astronomical Society. - 1538-4357 .- 0004-637X. ; 925:1
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Tidskriftsartikel (refereegranskat)abstract
- The 2 mm Mapping Obscuration to Reionization with ALMA (MORA) Survey was designed to detect high-redshift (z greater than or similar to 4), massive, dusty star-forming galaxies (DSFGs). Here we present two likely high-redshift sources, identified in the survey, whose physical characteristics are consistent with a class of optical/near-infrared (OIR)-invisible DSFGs found elsewhere in the literature. We first perform a rigorous analysis of all available photometric data to fit spectral energy distributions and estimate redshifts before deriving physical properties based on our findings. Our results suggest the two galaxies, called MORA-5 and MORA-9, represent two extremes of the "OIR-dark" class of DSFGs. MORA-5 (z(phot) = 4.3(-1.3)(+1.5)) is a significantly more active starburst with a star formation rate (SFR) of 830(-190)(+340) M-circle dot yr(-1) compared to MORA-9 (z(phot) = 4.3(-1.0)(+1.3)), whose SFR is a modest 200(-60)(+250) M-circle dot yr(-1). Based on the stellar masses (M-star approximate to 10(10-11) M-circle dot), space density (n similar to (5 +/- 2) x 10(-6) Mpc(-3), which incorporates two other spectroscopically confirmed OIR-dark DSFGs in the MORA sample at z = 4.6 and z = 5.9), and gas depletion timescales (<1 Gyr) of these sources, we find evidence supporting the theory that OIR-dark DSFGs are the progenitors of recently discovered 3 < z < 4 massive quiescent galaxies.
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| 3. |
- Asami, Jinta, et al.
(författare)
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Structural basis of hepatitis B virus receptor binding
- 2024
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Ingår i: Nature Structural & Molecular Biology. - 1545-9993 .- 1545-9985. ; 31, s. 447-454
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Tidskriftsartikel (refereegranskat)abstract
- 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.
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| 4. |
- Drew, David, 1976-
(författare)
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GFP as a tool to monitor membrane protein topology and overexpression in Escherichia coli
- 2005
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Membrane proteins are essential for life, and roughly one-quarter of all open reading frames in sequenced genomes code for membrane proteins. Unfortunately, our understanding of membrane proteins lags behind that of soluble proteins, and is best reflected by the fact that only 0.5% of the structures deposited in the protein data-bank (PDB) are of membrane proteins. This discrepancy has arisen because their hydrophobicity - which enables them to exist in a lipid environment - has made them resistant to most traditional approaches used for procuring knowledge from their soluble counter-parts. As such, novel methods are required to facilitate our knowledge acquisition of membrane proteins. In this thesis a generic approach for rapidly obtaining information on membrane proteins from the classic bacterial encyclopedia Escherichia coli is described. We have developed a Green Fluorescent Protein C-terminal tagging approach, with which we can acquire information as to the topology and ‘expressibility’ of membrane proteins in a high-throughput manner. This technology has been applied to the whole E. coli inner membrane proteome, and stands as an important advance for further membrane protein research.
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| 5. |
- Drew, David, 1976-, et al.
(författare)
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Ion and lipid orchestration of secondary active transport
- 2024
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Ingår i: Nature. - 0028-0836 .- 1476-4687. ; 626:8001, s. 963-974
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Forskningsöversikt (refereegranskat)abstract
- 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.
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| 6. |
- Mazza, Tiziano, et al.
(författare)
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Structural basis for the modulation of MRP2 activity by phosphorylation and drugs
- 2024
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Ingår i: Nature Communications. - 2041-1723. ; 15:1
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Tidskriftsartikel (refereegranskat)abstract
- 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.
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| 7. |
- Yeo, Hyunku, 1983-, et al.
(författare)
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Structure and electromechanical coupling of a voltage-gated Na+/H+ exchanger
- 2023
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Ingår i: Nature. - 0028-0836 .- 1476-4687. ; 623:7985, s. 193-201
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Tidskriftsartikel (refereegranskat)abstract
- 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.
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