Crystallizing Membrane Proteins in the Lipidic Mesophase. Experience with Human Prostaglandin E2 Synthase 1 and an Evolving Strategy

Abstract

The lipidic mesophase or in meso method for crystallizing membrane proteins has several high profile targets to its credit and is growing in popularity. Despite its success, the method is in its infancy as far as rational crystallogenesis is concerned. Consequently, significant time, effort, and resources are still required to generate structure-grade crystals, especially with a new target type. Therefore, a need exists for crystallogenesis protocols that are effective with a broad range of membrane protein types. Recently, a strategy for crystallizing a prokaryotic α-helical membrane protein, diacylglycerol kinase (DgkA), by the in meso method was reported (Cryst. Growth. Des.20131328462857). Here, we describe its application to the human α-helical microsomal prostaglandin E2 synthase 1 (mPGES1). While the DgkA strategy proved useful, significant modifications were needed to generate structure-quality crystals of this important therapeutic target. These included protein engineering, using an additive phospholipid in the hosting mesophase, performing multiple rounds of salt screening, and carrying out trials at 4 °C in the presence of a tight binding ligand. The crystallization strategy detailed here should prove useful for generating structures of other integral membrane proteins by the in meso method.

A dynamic Asp–Arg interaction is essential for catalysis in microsomal prostaglandin E 2 synthase

“…Furthermore, Arg-126 and Asp-49 participate in a charge interaction that could also contribute to catalysis. This structural information is supported by a mesophase crystal structure of an engineered version of mPGES-1 (14) and several inhibitor complexes that have recently been published (15).…”

Perspective of microsomal prostaglandin E2 synthase-1 as drug target in inflammation-related disorders

“…The rigid structure of mPGES-1 with interhelix hydrogen bonds might prevent the required comprehensive conformational changes [31]. Crystal structures of mPGES-1 in complex with an inhibitor were not available until recently, when mPGES-1 was cocrystalized with the high-affinity ligands BI1 and BI2 [38], a 2,4-biarylimidazole, the phenanthrene imidazole MF-63, a MK-886 analogue and an indole-2-carboxylic acid [39].…”

Protein crystallization for drug design in the last 50 years

“…The problems arise when the protein is unhappy in the detergent. The in meso method for crystallizing membrane proteins is growing in popularity (Li et al, 2014).…”