Huliciak M.,Palacky University |
Huliciak M.,University of Aarhus |
Huliciak M.,Center for Membrane Pumps in Cells and Disease kin |
Reinhard L.,University of Aarhus |
And 10 more authors.
Biochemical Pharmacology | Year: 2014
Cisplatin is the most widely used chemotherapeutics for cancer treatment, however, its administration is connected to inevitable adverse effects. Previous studies suggested that cisplatin is able to inhibit Na+/K+-ATPase (NKA), the enzyme responsible for maintaining electrochemical potential and sodium gradient across the plasma membrane. Here we report a crystallographic analysis of cisplatin bound to NKA in the ouabain bound E2P form. Despite a moderate resolution (7.4 A˚ and 7.9 A˚), the anomalous scattering from platinum and a model representation from a recently published structure enabled localization of seven cisplatin binding sites by anomalous difference Fourier maps. Comparison with NKA structures in the E1P conformation suggested two possible inhibitory mechanisms for cisplatin. Binding to Met151 can block the N-terminal pathway for transported cations, while binding to Met171 can hinder the interaction of cytoplasmic domains during the catalytic cycle. © 2014 Elsevier Inc. All rights reserved.
Autzen H.E.,Center for Membrane Pumps in Cells and Disease kin |
Autzen H.E.,University of Aarhus |
Siuda I.,Center for Membrane Pumps in Cells and Disease kin |
Siuda I.,Bioinformatics Research Center |
And 8 more authors.
Molecular Membrane Biology | Year: 2015
Like other integral membrane proteins, the activity of the Sarco/Endoplasmic Reticulum Ca2+-ATPase (SERCA) is regulated by the membrane environment. Cholesterol is present in the endoplasmic reticulum membrane at low levels, and it has the potential to affect SERCA activity both through direct, specific interaction with the protein or through indirect interaction through changes of the overall membrane properties. There are experimental data arguing for both modes of action for a cholesterol-mediated regulation of SERCA. In the current study, coarse-grained molecular dynamics simulations are used to address how a mixed lipid-cholesterol membrane interacts with SERCA. Candidates for direct regulatory sites with specific cholesterol binding modes are extracted from the simulations. The binding pocket for thapsigargin, a nanomolar inhibitor of SERCA, has been suggested as a cholesterol binding site. However, the thapsigargin binding pocket displayed very little cholesterol occupation in the simulations. Neither did atomistic simulations of cholesterol in the thapsigargin binding pocket support any specific interaction. The current study points to a non-specific effect of cholesterol on SERCA activity, and offers an alternative interpretation of the experimental results used to argue for a specific effect. © 2015 © 2015 Taylor & Francis.
De Ford C.,Albert Ludwigs University of Freiburg |
Calderon C.,University of Costa Rica |
Sehgal P.,University of Aarhus |
Sehgal P.,Center for Membrane Pumps in Cells and Disease kin |
And 10 more authors.
Journal of Natural Products | Year: 2015
Tricyclic clerodane diterpenes (TCDs) are natural compounds that often show potent cytotoxicity for cancer cells, but their mode of action remains elusive. A computationally based similarity search (CDRUG), combined with principal component analysis (ChemGPS-NP) and docking calculations (GOLD 5.2), suggested TCDs to be inhibitors of the sarco/endoplasmic reticulum Ca2+-ATPase (SERCA) pump, which is also the target of the sesquiterpene lactone thapsigargin. Biochemical studies were performed with 11 TCDs on purified rabbit skeletal muscle sarcoplasmic reticulum membranes, which are highly enriched with the SERCA1a isoform. Casearborin D (2) exhibited the highest affinity, with a KD value of 2 μM and giving rise to complete inhibition of SERCA1a activity. Structure-activity relationships revealed that functionalization of two acyl side chains (R1 and R4) and the hydrophobicity imparted by the aliphatic chain at C-9, as well as a C-3,C-4 double bond, play crucial roles for inhibitory activity. Docking studies also suggested that hydrophobic interactions in the binding site, especially with Phe256 and Phe834, may be important for a strong inhibitory activity of the TCDs. In conclusion, a novel class of SERCA inhibitory compounds is presented. © 2015 The American Chemical Society and American Society of Pharmacognosy.
PubMed | University of Aarhus and Center for Membrane Pumps in Cells and Disease kin
Type: | Journal: Methods in molecular biology (Clifton, N.J.) | Year: 2015
Nanodiscs are disc-shaped self-assembled lipid bilayers encircled by membrane scaffolding proteins derived from Apolipoprotein A-1 (apo A-1). They constitute a versatile tool for studying membrane proteins since reconstitution into nanodiscs allows studies of the membrane proteins in detergent-free aqueous solutions in a lipid bilayer. Here, we apply the technique to the Na(+),K(+)-ATPase (NKA) from pig kidney using Membrane Scaffolding Protein 1 D1 (MSP1D1). Contrary to other reports, the nanodiscs obtained by our protocol are built up of the native lipids originally present in the detergent solubilized sample together with the NKA.
Drachmann N.D.,Center for Membrane Pumps in Cells and Disease kin |
Olesen C.,Center for Membrane Pumps in Cells and Disease kin |
Moller J.V.,Center for Membrane Pumps in Cells and Disease kin |
Guo Z.,Center for Membrane Pumps in Cells and Disease kin |
And 2 more authors.
The FEBS journal | Year: 2014
The activity of the sarco/endoplasmic reticulum Ca(2+) -ATPase (SERCA) depends strongly on the lipid composition of the surrounding membrane. Yet, structural information on SERCA-lipid interaction is still relatively scarce, and the influence of different lipids on the enzyme is not well understood. We have analyzed SERCA crystal structures in the presence of four different phosphatidylcholine lipids of different lengths and double-bond compositions, and we find three different binding sites for lipid head groups, which are apparently independent of the acyl moiety of the lipids used. By comparison with other available SERCA structures with bound lipids, we find a total of five recurring sites, two of which are specific to certain conformational states of the enzyme, two others are state-independent, and one is a crucial site for crystal formation. Three of the binding sites overlap with or are in close vicinity to known binding sites for various SERCA-specific inhibitors and regulators, e.g. thapsigargin, sarcolipin/phospholamban and cyclopiazonic acid. Whereas the transient sites are amenable to a transient, regulatory influence of lipid molecules, the state-independent sites probably provide a flexible anchoring of the protein in the fluid bilayer. © 2014 FEBS.
PubMed | Center for Membrane Pumps in Cells and Disease kin
Type: Journal Article | Journal: Science (New York, N.Y.) | Year: 2013
The Na(+), K(+)-adenosine triphosphatase (ATPase) maintains the electrochemical gradients of Na(+) and K(+) across the plasma membrane--a prerequisite for electrical excitability and secondary transport. Hitherto, structural information has been limited to K(+)-bound or ouabain-blocked forms. We present the crystal structure of a Na(+)-bound Na(+), K(+)-ATPase as determined at 4.3 resolution. Compared with the K(+)-bound form, large conformational changes are observed in the subunit whereas the and subunit structures are maintained. The locations of the three Na(+) sites are indicated with the unique site III at the recently suggested IIIb, as further supported by electrophysiological studies on leak currents. Extracellular release of the third Na(+) from IIIb through IIIa, followed by exchange of Na(+) for K(+) at sites I and II, is suggested.
PubMed | Center for Membrane Pumps in Cells and Disease kin
Type: Journal Article | Journal: Acta crystallographica. Section F, Structural biology communications | Year: 2016
Na(+),K(+)-ATPase is responsible for the transport of Na(+) and K(+) across the plasma membrane in animal cells, thereby sustaining vital electrochemical gradients that energize channels and secondary transporters. The crystal structure of Na(+),K(+)-ATPase has previously been elucidated using the enzyme from native sources such as porcine kidney and shark rectal gland. Here, the isolation, crystallization and first structure determination of bovine kidney Na(+),K(+)-ATPase in a high-affinity E2-BeF3(-)-ouabain complex with bound magnesium are described. Crystals belonging to the orthorhombic space group C2221 with one molecule in the asymmetric unit exhibited anisotropic diffraction to a resolution of 3.7 with full completeness to a resolution of 4.2. The structure was determined by molecular replacement, revealing unbiased electron-density features for bound BeF3(-), ouabain and Mg(2+) ions.