Solution NMR Structure of Diacylglycerol Kinase (DAGK), A 40 kDa Integral Membrane Protein

Abstract

Membrane proteins which comprise about 1/3 of the proteome play very important physiological roles in the cell. However compared to soluble counterparts, relatively feww membrane protein structures have been determined by any method. Structure determination of multi-span alpha-helical integral membrane proteins by NMR has been limited by the relatively poor spectral deispersion associated with their helical nature and difficulty in obtaining long-range distance restraints. Escherichia coli DAGK is a 40 kDa homotrimeric membrane protein composed of 121 residues. Each subunit has three transmembrane helices. DAGK catalyzes the phosphorylation of the lipid diacylglycerol by MgATP to phosphatidic acid and MgADP, and has served as a model system for membrane protein biocatalysis, stability and folding. To determine the structure of DAGK in detergent micelles, we have employed traditional chemical shift and NOE restraints, as well as paramagnetic relaxation enhancement (PRE) to obtain long-range distances. Here we present the NMR-derived structure of E. coli DAGK using long-range restraints obtained from paramagnetic relaxation enhancement combined with traditional restraints.