AF2 of Stable-5-LOX and variants

In the absence of structural models and, despite extensive crystallization trials for the kinetically faster variants of Stable-5-LOX, we considered computational experiments to fill the gap. We utilized the structure prediction method AlphaFold2 (AF2), which showed predictive scores near atomic accuracy as demonstrated in the 14th Critical Assessment of protein Structure Prediction competition (CASP14). Subsequently, the ColabFold notebook was released, which allows novice users an opportunity to predict protein structures in the AF2 environment coupled through the Google Collaboratory cloud services. We utilized the advanced version of AF2 where no homology templates were allowed. We generated ten predicted models for each variant with all models scoring above 90 in the predicted local distance difference test (pLDDT). As expected, the structures of the membrane-binding loops on the PLAT domain diverged more than the body of the protein with some of the lowest pLDDT scores in these regions. Notably, structural divergence in the helix alpha 2 region was consistently the highest with the lowest pLDDT scores. We superimposed all predicted models of the various mutants with the closed structure of Stable-5-LOX (PDB code: 3O8Y) to get an all atom r.m.s.d. given in angstrom. We then visually inspected if helix alpha 2 was in an elongated form and whether F177 and Y181 were plugging the active site closed.