New crystal structure helps explain 50 years of research on a novel membrane enzyme in E. coli

All cells have a cell membrane, made up of a lipid bilayer. Through this membrane sit the membrane proteins; a vital family of proteins which control how the cell interacts with its environment, and control passage of materials in and out of the cell. The family are challenging to study, as the process of crystallisation – the first step to determining a protein’s atomic structure – is incredibly difficult to perform with lipid soluble proteins. Researchers from Trinity College Dublin have now discovered the structure of the membrane protein, diacylglyerol kinase, DgkA, an enzyme found in bacterial cell membranes, using beamline I24 at Diamond Light Source.

Diacylglycerol kinase is an integral membrane enzyme involved in the synthesis of key components of the cellular envelope, including lipopolysaccharide, in Gram-negative bacteria. The diminutive enzyme, the smallest known kinase, uses two very different substrates. Its lipid substrate resides in the fatty membrane that surrounds the cell, while its co-substrate, known as ATP, is completely water soluble. Having the crystal structure of the enzyme helps explain how DgkA is able to bring together substrates with such different qualities. These findings are notable as they help to explain almost half a century of investigations, and reveal profound differences between the crystal structure and the structure which had been previously obtained using nuclear magnetic resonance (NMR).

Professor Martin Caffrey, head of the Membrane Structural and Functional Biology Group in Trinity's School of Medicine and School of Biochemistry and Immunology, praised the work of Dr Dianfan Li, the research associate who led the study. “He went from a synthetic gene to a crystal very quickly – within one week.” said Professor Caffrey. “It then took us three years to get the structure! This was a huge amount of work, a lot of which was done in a 4 degree refrigerator as this is the only place where the crystals would grow. That's not a particularly comfortable place to work.”

For 50 years, DGKA has served as a paradigm for investigating membrane protein enzymology, folding, assembly and stability. To understand how the enzyme functions at a molecular level, the team has determined the structure of DgkA from Escherichia coli (E. coli) using X-rays from Diamond Light Source and the Advanced Photon Source (APS) synchrotrons. These findings will rationalise the extensive published biochemical and biophysical data on this novel kinase.

The lipid and ATP substrates are forced into a molecular embrace at the cytoplasm interface. Residing just atomic distances apart, and perfectly aligned for reaction in the maw of the enzyme, the two molecules are thought to fuse, ever so briefly, to become a single entity. In a series of follow-up enzyme reactions, the newly formed phospholipid contributes to cell envelope synthesis.

“We collected the data using the I24 [beamline], which is a fantastic instrument. It provided us with a laser-like beam, allowing us to hunt for the crystals within an opaque mesophase. Understanding these structures, and the membrane proteins and their functionality, can lead to new drug discovery and further development of existing drugs.” Professor Caffrey. Trinity College Dublin.

Crystal structure of the integral membrane diacylglycerol kinase

Dianfan Li, Joseph A. Lyons, Valerie E. Pye, Lutz Vogeley, David Aragão, Colin P. Kenyon, Syed T. A. Shah, Christine Doherty, Margaret Aherne & Martin Caffrey

Nature (2013)

Doi:10.1038/nature12179

Published online15 May 2013

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New crystal structure helps explain 50 years of research on a novel membrane enzyme in E. coli

New crystal structure helps explain 50 years of research on a novel membrane enzyme in E. coli