Prof. Inga Schmidt-Krey, GIT

Structural Analysis of Human Leukotriene C4 Synthase by Electron Crystallography

The membrane protein human leukotriene C4 synthase (LTC4S) is a potential drug target in asthma, due to its role of catalyzing the conjugation of leukotriene A4 to reduced glutathione to produce leukotriene C4 (LTC4). LTC4 and its metabolites LTD4 and LTE4 are responsible for the characteristic symptoms of asthma including bronchoconstriction, mucus hypersecretion and oedema of the airways. The mostly hydrophobic nature of LTC4S makes it an ideal specimen for structural studies by two-dimensional (2D) crystallization and electron crystallography.

Human LTC4S was overexpressed in and purified from Schizosaccharomyces pombe and induced to form 2D crystals through detergent removal by dialysis. Crystals were embedded in 4.5% trehalose and frozen by back-injection. Spot-scan images of these specimens were collected with a JEOL 3000 SFF electron microscope at an accelerating voltage of 300kV and a specimen temperature of 4K at a nominal magnification of 70,000. Images were then selected by optical diffraction, scanned with a Zeiss SCAI scanner, and processed with the MRC programs.
The projection data at a resolution of 4.5Å showed the crystals to have p321 symmetry with unit cell dimensions of a=b=73.4 Å, ϒ=120º. Furthermore, the projection map clearly showed a trimer. When these data were truncated in the intermediate resolution range at 7.5 Å, densities corresponding to four transmembrane α-helices per LTC4 synthase monomer could be identified. This agrees well with biochemical data placing residues of the active site in two loops on the cytoplasmic side of the membrane.

Currently we are collecting electron cryo-microscopy three-dimensional data for an atomic model to ultimately be able to elucidate the molecular mechanism of LTC4S.

For more information contract Dr. Andrew Lyon (404-894-4090).