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.

Product formation controlled by substrate dynamics in leukotriene A4 hydrolase

“…Leukotriene biosynthesis starts with the catalytic action of 5LO [48]. Monomeric 5LO is the active form and binds to the membrane surface of nanodiscs in a productive manner in terms of product formation [45]. As mentioned earlier (the above paragraph and in section 1.3.1), 5LO requires another protein called FLAP for efficient catalysis.…”

“…Liposomes are better membrane mimics than detergent. Nevertheless, it would be very difficult to visualize the same sample of a protein-membrane-complex by both native PAGE and TEM which were mainly used in this thesis [45,116]. Liposomes can be made of a small size, however, the curvature increases which may be a disadvantage.…”

“…This orientation of dimeric 5LO might hinder the substrate access portal leading to reduced production of LTA 4 . The dimeric 5LO lacks enzymatic activity and cannot bind to a membrane in vitro [45].…”

“…The TEM analysis showed different orientations of dimeric 5LO. The dimer formation was prevented by using a reducing agent TCEP in all the working solution [45].…”

Nanodiscs in Membrane Biochemistry and Biophysics

“…211 Helices rearrange in the membrane with inter-helical loops forming a funnel for the entrance to the pore. Others have utilized Nanodiscs in electron microscopic investigations of lipoxygenase, 212 drug efflux pumps, 213 the magnesium channel, 214 AMP dependent protein kinase, 93 the ribosome-SecYE complex 215 and other membrane proteins. 119,216 Thus Nanodiscs and electron microscopy have become a mainstay for the structural determination of membrane proteins and their interactions to form supramolecular complexes involved in signaling, energy transduction and transport.…”