Structural Dynamics of 15-Lipoxygenase-2 via Hydrogen Deuterium Exchange.

Eicosanoids are inflammatory signaling lipids that are biosynthesized in response to cellular injury or threat. They were originally thought to be pro-inflammatory molecules, but members of at least one subclass, the lipoxins, are able to resolve inflammation. One step in lipoxin synthesis is the oxygenation of arachidonic acid by 15-lipoxygenase LOX). 15-LOX contains two domains: a Ca2+. binding PLAT domain and a catalytic domain. 15-LOX is a soluble cytosolic protein until binding of Ca2+ to the PLAT domain promotes translocation to the membrane surfac. The role of 15-LOX structural dynamics in this translocation has remained:unclear. We investigated the dynamics of 15-LOX isoform 13 (15-LOX-2) upon binding of Ca2+ and ligands, as well as upon membrane association using hydrogen deuterium exchange mass spectrometry (HDX-MS). We used HDX-MS to probe the solvent accessibility and backbone flexibility of 15-LOX-2, revealing significant differences in deuterium incorporation between the PLAT and catalytic domains, with the PLAT domain demonstrating higher flexibility. Comparison of HDX for 15-LOX-2 in the presence and absence of Ca2+ indicates there are few differences in structural dynamics. Furthermore,,our HDX results involving nanodisc-associated 15-LOX-2 suggest that significant structural and dynamic changes in 15-LOX-2 are not required for membrane association. Our results also show that a substrate lipid binding to the active site in the catalytic domain does induce changes in incorporation of deuterium into the PLAT dornain.-Overall, our results challenge the previous hypothesis that Ca2+ binding induces major structural changes in the PLAT domain and support the hypothesis that is interdorriain communication in 15-LOX-2.