Crystallization and structure solution of phospholipaseacyltransferase in complex with potent inhibitors

K.C. Zwiers, S. Chatterjee, P. Voskamp, J. Zhou, K. Al Ayed, B. Narhe, M. Ubbink, M. van der Stelt, N.S. Pannu

Gorlaeus Laboratories, Leiden institute of Chemistry, Leiden University, The Netherlands.

k.c.zwiers@umail.leidenuniv.nl

 

Over one third of adults in America are obese and thus are at risk on type 2 diabetes, cancer and cardiovascular diseases. Phospolipase-Acyltranferase (PLA/AT) are important enzymes that are crucial in the development of obesity. We have recently designed a class of reversible PLA/AT inhibitors showing micromolar inhibition that are promising candidate anti-cancer or anti-obese drugs. The crystal structure of PLA1/AT3 and PLA2/AT3 have been reported [1], but the binding pocket is very large and consequently the inhibitors may have multiple binding modes, Here, we describe the crystallization and preliminary structure analysis if PLA/AT soaked with various inhibitors. Soaking PLA/AT protein crystals with inhibitors remains challenging, as the designed inhibitors are poorly soluble. Our results demonstrate that it was possible to obtain inhibitor soaked non-damaged crystals for PLA/AT 2, by varying the concentrations of inhibitor and various solubilizing agents, cryo protectant and protein crystal buffer solution. The resulting crystals were collected and resulted in high-resolution datasets of 1.4 Ă… respectively. We will optimize these results in future co-crystallization experiments or for the improvement of soaking other PLA/AT family members (1, 3-5) with a wide variety of inhibitors.

[1] M. Golczak, A. E. Sears, P. D. Kiser, and K. Palczewski, “LRAT-specific domain facilitates vitamin A metabolism by domain swapping in HRASLS3, Nat. Chem. Biol., vol. 11, no. 1, pp. 26-32, Jan. 2015.