title: Structural and Functional Analysis of Calcium Ion Mediated Binding of 5-Lipoxygenase to Nanodiscs.
abstract: An important step in the production of inflammatory mediators of the leukotriene family is the Ca2+ mediated recruitment of 5 Lipoxygenase (5LO) to nuclear membranes. To study this reaction in vitro, the natural membrane mimicking environment of nanodiscs was used. Nanodiscs with 10.5 nm inner diameter were made with the lipid POPC and membrane scaffolding protein MSP1E3D1. Monomeric and dimeric 5LO were investigated. Monomeric 5LO mixed with Ca2+ and nanodiscs are shown to form stable complexes that 1) produce the expected leukotriene products from arachidonic acid and 2) can be, for the first time, visualised by native gel electrophoresis and negative stain transmission electron microscopy and 3) show a highest ratio of two 5LO per nanodisc. We interpret this as one 5LO on each side of the disc. The dimer of 5LO is visualised by negative stain transmission electron microscopy and is shown to not bind to nanodiscs. This study shows the advantages of nanodiscs to obtain basic structural information as well as functional information of a complex between a monotopic membrane protein and the membrane.
authors.affiliation: Department of Biosciences and Nutrition, Karolinska Institutet, 14183 Huddinge, Sweden.|School of Technology and Health, KTH Royal Institute of Technology, 14183 Huddinge, Sweden.
authors.forename: Ramakrishnan B
authors.initials: RB
authors.lastname: Kumar
authors.affiliation: Department of Biosciences and Nutrition, Karolinska Institutet, 14183 Huddinge, Sweden.|School of Technology and Health, KTH Royal Institute of Technology, 14183 Huddinge, Sweden.
authors.forename: Lin
authors.initials: L
authors.lastname: Zhu
authors.affiliation: Rheumatology Unit, Department of Medicine, Karolinska Institutet, 17176 Stockholm, Sweden.
authors.forename: Helena
authors.initials: H
authors.lastname: Idborg
authors.affiliation: Division of Physiological Chemistry II, Department of Medical Biochemistry and Biophysics, Karolinska Institutet, 17165 Stockholm, Sweden.
authors.forename: Olof
authors.initials: O
authors.lastname: Rådmark
authors.affiliation: Rheumatology Unit, Department of Medicine, Karolinska Institutet, 17176 Stockholm, Sweden.
authors.forename: Per-Johan
authors.initials: PJ
authors.lastname: Jakobsson
authors.affiliation: Division of Physiological Chemistry II, Department of Medical Biochemistry and Biophysics, Karolinska Institutet, 17165 Stockholm, Sweden.
authors.forename: Agnes
authors.initials: A
authors.lastname: Rinaldo-Matthis
authors.affiliation: Department of Biosciences and Nutrition, Karolinska Institutet, 14183 Huddinge, Sweden.|School of Technology and Health, KTH Royal Institute of Technology, 14183 Huddinge, Sweden.
authors.forename: Hans
authors.initials: H
authors.lastname: Hebert
authors.affiliation: Department of Biosciences and Nutrition, Karolinska Institutet, 14183 Huddinge, Sweden.|School of Technology and Health, KTH Royal Institute of Technology, 14183 Huddinge, Sweden.
authors.forename: Caroline
authors.initials: C
authors.lastname: Jegerschöld
mesh_terms: D001094:Arachidonate 5-Lipoxygenase / Q000737:chemistry / Q000378:metabolism*; D002118:Calcium / Q000378:metabolism*; D002850:Chromatography, Gel; D019281:Dimerization; D006801:Humans; D046529:Microscopy, Electron, Transmission; D049329:Nanostructures; D063347:Native Polyacrylamide Gel Electrophoresis; D011485:Protein Binding
chemical_list: D001094:Arachidonate 5-Lipoxygenase; D002118:Calcium