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

DOI: 10.1021/cg500157x DOI Help

Authors: Dianfan Li (Trinity College, Dublin) , Nicole Howe (Trinity College, Dublin) , Abhiram Dukkipati (Trinity College, Dublin) , Syed T. A. Shah (Trinity College, Dublin) , Benjamin D. Bax (GlaxoSmithKline, Medicines Research Centre) , Colin Edge (GlaxoSmithKline, Medicines Research Centre) , Angela Bridges (GlaxoSmithKline, Medicines Research Centre) , Phil Hardwicke (GlaxoSmithKline, Medicines Research Centre) , Onkar M. P. Singh (GlaxoSmithKline, Medicines Research Centre) , Ged Giblin (GlaxoSmithKline, Medicines Research Centre) , Alexander Pautsch (Boehringer Ingelheim Pharma GmbH) , Roland Pfau (Boehringer Ingelheim Pharma GmbH) , Gisela Schnapp (Boehringer Ingelheim Pharma GmbH) , Meitian Wang (Swiss Light Source, Paul Scherrer Institute) , Vincent Olieric (Swiss Light Source, Paul Scherrer Institute) , Martin Caffrey (Trinity College, Dublin)
Co-authored by industrial partner: Yes

Type: Journal Paper
Journal: Crystal Growth & Design , VOL 14 (4)

State: Published (Approved)
Published: March 2014
Diamond Proposal Number(s): 8224

Open Access Open Access

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 ?-helical membrane protein, diacylglycerol kinase (DgkA), by the in meso method was reported ( Cryst. Growth. Des. 2013, 13, 2846?2857). Here, we describe its application to the human ?-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.

Journal Keywords: Human Prostaglandin E2 Synthase; Lipidic Mesophase

Subject Areas: Biology and Bio-materials

Instruments: I24-Microfocus Macromolecular Crystallography

Other Facilities: Swiss Light Source (SLS), Advanced Photon Source (APS)