Publications by authors named "Bjørn Panyella Pedersen"

12 Publications

  • Page 1 of 1

An Intracellular Pathway Controlled by the N-terminus of the Pump Subunit Inhibits the Bacterial KdpFABC Ion Pump in High K Conditions.

J Mol Biol 2021 May 2;433(15):167008. Epub 2021 May 2.

PHYLIFE: Physical Life Science, Department of Physics, Chemistry and Pharmacy, University of Southern Denmark, Campusvej 55, Odense 5230 M, Denmark. Electronic address:

The heterotetrameric bacterial KdpFABC transmembrane protein complex is an ion channel-pump hybrid that consumes ATP to import K against its transmembrane chemical potential gradient in low external K environments. The KdpB ion-pump subunit of KdpFABC is a P-type ATPase, and catalyses ATP hydrolysis. Under high external K conditions, K can diffuse into the cells through passive ion channels. KdpFABC must therefore be inhibited in high K conditions to conserve cellular ATP. Inhibition is thought to occur via unusual phosphorylation of residue Ser162 of the TGES motif of the cytoplasmic A domain. It is proposed that phosphorylation most likely traps KdpB in an inactive E1-P like conformation, but the molecular mechanism of phosphorylation-mediated inhibition remains unknown. Here, we employ molecular dynamics (MD) simulations of the dephosphorylated and phosphorylated versions of KdpFABC to demonstrate that phosphorylated KdpB is trapped in a conformation where the ion-binding site is hydrated by an intracellular pathway between transmembrane helices M1 and M2 which opens in response to the rearrangement of cytoplasmic domains resulting from phosphorylation. Cytoplasmic access of water to the ion-binding site is accompanied by a remarkable loss of secondary structure of the KdpB N-terminus and disruption of a key salt bridge between Glu87 in the A domain and Arg212 in the P domain. Our results provide the molecular basis of a unique mechanism of regulation amongst P-type ATPases, and suggest that the N-terminus has a significant role to play in the conformational cycle and regulation of KdpFABC.
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http://dx.doi.org/10.1016/j.jmb.2021.167008DOI Listing
May 2021

Structural comparison of GLUT1 to GLUT3 reveal transport regulation mechanism in sugar porter family.

Life Sci Alliance 2021 04 3;4(4). Epub 2021 Feb 3.

Department of Molecular Biology and Genetics, Aarhus University, Aarhus C, Denmark

The human glucose transporters GLUT1 and GLUT3 have a central role in glucose uptake as canonical members of the Sugar Porter (SP) family. GLUT1 and GLUT3 share a fully conserved substrate-binding site with identical substrate coordination, but differ significantly in transport affinity in line with their physiological function. Here, we present a 2.4 Å crystal structure of GLUT1 in an inward open conformation and compare it with GLUT3 using both structural and functional data. Our work shows that interactions between a cytosolic "SP motif" and a conserved "A motif" stabilize the outward conformational state and increases substrate apparent affinity. Furthermore, we identify a previously undescribed Cl ion site in GLUT1 and an endofacial lipid/glucose binding site which modulate GLUT kinetics. The results provide a possible explanation for the difference between GLUT1 and GLUT3 glucose affinity, imply a general model for the kinetic regulation in GLUTs and suggest a physiological function for the defining SP sequence motif in the SP family.
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http://dx.doi.org/10.26508/lsa.202000858DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7898563PMC
April 2021

Mechanistic Insight into Lipid Binding to Yeast Niemann Pick Type C2 Protein.

Biochemistry 2020 11 3;59(45):4407-4420. Epub 2020 Nov 3.

Department of Biochemistry and Molecular Biology, University of Southern Denmark, Campusvej 55, DK-5230 Odense M, Denmark.

Niemann Pick type C2 (NPC2) is a small sterol binding protein in the lumen of late endosomes and lysosomes. We showed recently that the yeast homologue of NPC2 together with its binding partner NCR1 mediates integration of ergosterol, the main sterol in yeast, into the vacuolar membrane. Here, we study the binding specificity and the molecular details of lipid binding to yeast NPC2. We find that NPC2 binds fluorescence- and spin-labeled analogues of phosphatidylcholine (PC), phosphatidylserine, phosphatidylinositol (PI), and sphingomyelin. Spectroscopic experiments show that NPC2 binds lipid monomers in solution but can also interact with lipid analogues in membranes. We further identify ergosterol, PC, and PI as endogenous NPC2 ligands. Using molecular dynamics simulations, we show that NPC2's binding pocket can adapt to the ligand shape and closes around bound ergosterol. Hydrophobic interactions stabilize the binding of ergosterol, but binding of phospholipids is additionally stabilized by electrostatic interactions at the mouth of the binding site. Our work identifies key residues that are important in stabilizing the binding of a phospholipid to yeast NPC2, thereby rationalizing future mutagenesis studies. Our results suggest that yeast NPC2 functions as a general "lipid solubilizer" and binds a variety of amphiphilic lipid ligands, possibly to prevent lipid micelle formation inside the vacuole.
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http://dx.doi.org/10.1021/acs.biochem.0c00574DOI Listing
November 2020

Structural Insight into Eukaryotic Sterol Transport through Niemann-Pick Type C Proteins.

Cell 2019 10 19;179(2):485-497.e18. Epub 2019 Sep 19.

Department of Molecular Biology and Genetics, Aarhus University, Gustav Wieds Vej 10, Aarhus C 8000, Denmark; Aarhus Institute of Advanced Studies, Aarhus University, Høegh-Guldbergs Gade 6B, Aarhus C 8000, Denmark. Electronic address:

Niemann-Pick type C (NPC) proteins are essential for sterol homeostasis, believed to drive sterol integration into the lysosomal membrane before redistribution to other cellular membranes. Here, using a combination of crystallography, cryo-electron microscopy, and biochemical and in vivo studies on the Saccharomyces cerevisiae NPC system (NCR1 and NPC2), we present a framework for sterol membrane integration. Sterols are transferred between hydrophobic pockets of vacuolar NPC2 and membrane-protein NCR1. NCR1 has its N-terminal domain (NTD) positioned to deliver a sterol to a tunnel connecting NTD to the luminal membrane leaflet 50 Å away. A sterol is caught inside this tunnel during transport, and a proton-relay network of charged residues in the transmembrane region is linked to this tunnel supporting a proton-driven transport mechanism. We propose a model for sterol integration that clarifies the role of NPC proteins in this essential eukaryotic pathway and that rationalizes mutations in patients with Niemann-Pick disease type C.
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http://dx.doi.org/10.1016/j.cell.2019.08.038DOI Listing
October 2019

- automatic molecular dynamics flexible fitting of structural models into cryo-EM and crystallography experimental maps.

IUCrJ 2019 Jul 27;6(Pt 4):526-531. Epub 2019 Jun 27.

Centre for Structural Biology, Department of Molecular Biology and Genetics, Aarhus University, Gustav Wieds Vej 10C, Aarhus, DK-8000, Denmark.

Model building into experimental maps is a key element of structural biology, but can be both time consuming and error prone for low-resolution maps. Here we present , an easy-to-use tool that enables the user to run a molecular dynamics flexible fitting simulation followed by real-space refinement in an automated manner through a pipeline system. will modify an atomic model to fit within cryo-EM or crystallography density maps, and can be used advantageously for both the initial fitting of models, and for a geometrical optimization step to correct outliers, clashes and other model problems. We have benchmarked against 39 deposited cryo-EM models and maps, and observe model improvements in 34 of these cases (87%). Clashes between atoms were reduced, and the model-to-map fit and overall model geometry were improved, in several cases substantially. We show that is able to model large-scale conformational changes compared to the starting model. is a fast and easy tool for structural model builders at all skill levels. is available as a web service (https://namdinator.au.dk), or it can be run locally as a command-line tool.
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http://dx.doi.org/10.1107/S2052252519007619DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6608625PMC
July 2019

Crystal structure of the plant symporter STP10 illuminates sugar uptake mechanism in monosaccharide transporter superfamily.

Nat Commun 2019 01 24;10(1):407. Epub 2019 Jan 24.

Department of Molecular Biology and Genetics, Aarhus University, Gustav Wieds Vej 10, DK-8000, Aarhus C, Denmark.

Plants are dependent on controlled sugar uptake for correct organ development and sugar storage, and apoplastic sugar depletion is a defense strategy against microbial infections like rust and mildew. Uptake of glucose and other monosaccharides is mediated by Sugar Transport Proteins, proton-coupled symporters from the Monosaccharide Transporter (MST) superfamily. We present the 2.4 Å structure of Arabidopsis thaliana high affinity sugar transport protein, STP10, with glucose bound. The structure explains high affinity sugar recognition and suggests a proton donor/acceptor pair that links sugar transport to proton translocation. It contains a Lid domain, conserved in all STPs, that locks the mobile transmembrane domains through a disulfide bridge, and creates a protected environment which allows efficient coupling of the proton gradient to drive sugar uptake. The STP10 structure illuminates fundamental principles of sugar transport in the MST superfamily with implications for both plant antimicrobial defense, organ development and sugar storage.
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http://dx.doi.org/10.1038/s41467-018-08176-9DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6345825PMC
January 2019

Crystal structure of the potassium-importing KdpFABC membrane complex.

Nature 2017 06 21;546(7660):681-685. Epub 2017 Jun 21.

Department of Cell Biology, New York University School of Medicine, Skirball Institute, 540 First Avenue, New York, New York 10016, USA.

Cellular potassium import systems play a fundamental role in osmoregulation, pH homeostasis and membrane potential in all domains of life. In bacteria, the kdp operon encodes a four-subunit potassium pump that maintains intracellular homeostasis, cell shape and turgor under conditions in which potassium is limiting. This membrane complex, called KdpFABC, has one channel-like subunit (KdpA) belonging to the superfamily of potassium transporters and another pump-like subunit (KdpB) belonging to the superfamily of P-type ATPases. Although there is considerable structural and functional information about members of both superfamilies, the mechanism by which uphill potassium transport through KdpA is coupled with ATP hydrolysis by KdpB remains poorly understood. Here we report the 2.9 Å X-ray structure of the complete Escherichia coli KdpFABC complex with a potassium ion within the selectivity filter of KdpA and a water molecule at a canonical cation site in the transmembrane domain of KdpB. The structure also reveals two structural elements that appear to mediate the coupling between these two subunits. Specifically, a protein-embedded tunnel runs between these potassium and water sites and a helix controlling the cytoplasmic gate of KdpA is linked to the phosphorylation domain of KdpB. On the basis of these observations, we propose a mechanism that repurposes protein channel architecture for active transport across biomembranes.
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http://dx.doi.org/10.1038/nature22970DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5495170PMC
June 2017

Expression strategies for structural studies of eukaryotic membrane proteins.

Curr Opin Struct Biol 2016 06 27;38:137-44. Epub 2016 Jun 27.

Danish Research Institute of Translational Neuroscience (DANDRITE), Nordic EMBL Partnership for Molecular Medicine, Aarhus University, Aarhus, Denmark; Centre for Membrane Pumps in Cells and Disease-PUMPkin, Danish National Research Foundation, Aarhus, Denmark; Department of Molecular Biology and Genetics, Aarhus University, Aarhus, Denmark. Electronic address:

Integral membrane proteins in eukaryotes are central to various cellular processes and key targets in structural biology, biotechnology and drug development. However, the number of available structures for eukaryotic membrane protein belies their physiological importance. Recently, the number of available eukaryotic membrane protein structures has been steadily increasing due to the development of novel strategies in construct design, expression and structure determination. Here, we examine the major expression systems exploited for eukaryotic membrane proteins. Additionally we strive to tabulate and describe the recent expression strategies in eukaryotic membrane protein structural biology. We find that a majority of targets have been expressed in advanced host systems and modified from their wild-type form with distinct focus on conformation and thermostabilisation. However, strategies for native protein purification should also be considered where possible, particularly in light of the recent advances in single particle cryo electron microscopy.
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http://dx.doi.org/10.1016/j.sbi.2016.06.011DOI Listing
June 2016

Initiating heavy-atom-based phasing by multi-dimensional molecular replacement.

Acta Crystallogr D Struct Biol 2016 Mar 1;72(Pt 3):440-5. Epub 2016 Mar 1.

Centre for Membrane Pumps in Cells and Disease, Danish National Research Foundation, Department of Molecular Biology and Genetics, Aarhus University, Gustav Wieds Vej 10C, DK-8000 Aarhus, Denmark.

To obtain an electron-density map from a macromolecular crystal the phase problem needs to be solved, which often involves the use of heavy-atom derivative crystals and concomitant heavy-atom substructure determination. This is typically performed by dual-space methods, direct methods or Patterson-based approaches, which however may fail when only poorly diffracting derivative crystals are available. This is often the case for, for example, membrane proteins. Here, an approach for heavy-atom site identification based on a molecular-replacement parameter matrix (MRPM) is presented. It involves an n-dimensional search to test a wide spectrum of molecular-replacement parameters, such as different data sets and search models with different conformations. Results are scored by the ability to identify heavy-atom positions from anomalous difference Fourier maps. The strategy was successfully applied in the determination of a membrane-protein structure, the copper-transporting P-type ATPase CopA, when other methods had failed to determine the heavy-atom substructure. MRPM is well suited to proteins undergoing large conformational changes where multiple search models should be considered, and it enables the identification of weak but correct molecular-replacement solutions with maximum contrast to prime experimental phasing efforts.
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http://dx.doi.org/10.1107/S2059798315022482DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4784675PMC
March 2016

Structural basis for alternating access of a eukaryotic calcium/proton exchanger.

Nature 2013 Jul 19;499(7456):107-10. Epub 2013 May 19.

Department of Biochemistry and Biophysics, University of California, San Francisco, California 94158, USA.

Eukaryotic Ca(2+) regulation involves sequestration into intracellular organelles, and expeditious Ca(2+) release into the cytosol is a hallmark of key signalling transduction pathways. Bulk removal of Ca(2+) after such signalling events is accomplished by members of the Ca(2+):cation (CaCA) superfamily. The CaCA superfamily includes the Na(+)/Ca(2+) (NCX) and Ca(2+)/H(+) (CAX) antiporters, and in mammals the NCX and related proteins constitute families SLC8 and SLC24, and are responsible for the re-establishment of Ca(2+) resting potential in muscle cells, neuronal signalling and Ca(2+) reabsorption in the kidney. The CAX family members maintain cytosolic Ca(2+) homeostasis in plants and fungi during steep rises in intracellular Ca(2+) due to environmental changes, or following signal transduction caused by events such as hyperosmotic shock, hormone response and response to mating pheromones. The cytosol-facing conformations within the CaCA superfamily are unknown, and the transport mechanism remains speculative. Here we determine a crystal structure of the Saccharomyces cerevisiae vacuolar Ca(2+)/H(+) exchanger (Vcx1) at 2.3 Å resolution in a cytosol-facing, substrate-bound conformation. Vcx1 is the first structure, to our knowledge, within the CAX family, and it describes the key cytosol-facing conformation of the CaCA superfamily, providing the structural basis for a novel alternating access mechanism by which the CaCA superfamily performs high-throughput Ca(2+) transport across membranes.
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http://dx.doi.org/10.1038/nature12233DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3702627PMC
July 2013

Crystal structure of a copper-transporting PIB-type ATPase.

Nature 2011 Jun 29;475(7354):59-64. Epub 2011 Jun 29.

Centre for Membrane Pumps in Cells and Disease-PUMPKIN, Danish National Research Foundation, Aarhus University, Department of Molecular Biology, Gustav Wieds Vej 10C, DK-8000 Aarhus C, Denmark.

Heavy-metal homeostasis and detoxification is crucial for cell viability. P-type ATPases of the class IB (PIB) are essential in these processes, actively extruding heavy metals from the cytoplasm of cells. Here we present the structure of a PIB-ATPase, a Legionella pneumophila CopA Cu(+)-ATPase, in a copper-free form, as determined by X-ray crystallography at 3.2 Å resolution. The structure indicates a three-stage copper transport pathway involving several conserved residues. A PIB-specific transmembrane helix kinks at a double-glycine motif displaying an amphipathic helix that lines a putative copper entry point at the intracellular interface. Comparisons to Ca(2+)-ATPase suggest an ATPase-coupled copper release mechanism from the binding sites in the membrane via an extracellular exit site. The structure also provides a framework to analyse missense mutations in the human ATP7A and ATP7B proteins associated with Menkes' and Wilson's diseases.
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http://dx.doi.org/10.1038/nature10191DOI Listing
June 2011