Ternary structure reveals mechanism of a membrane diacylglycerol kinase

Abstract

Diacylglycerol kinase catalyses the ATP-dependent conversion of diacylglycerol to phosphatidic acid in the plasma membrane of Escherichia coli. The small size of this integral membrane trimer, which has 121 residues per subunit, means that available protein must be used economically to craft three catalytic and substrate-binding sites centred about the membrane/cytosol interface. How nature has accomplished this extraordinary feat is revealed here in a crystal structure of the kinase captured as a ternary complex with bound lipid substrate and an ATP analogue. Residues, identified as essential for activity by mutagenesis, decorate the active site and are rationalized by the ternary structure. The γ-phosphate of the ATP analogue is positioned for direct transfer to the primary hydroxyl of the lipid whose acyl chain is in the membrane. A catalytic mechanism for this unique enzyme is proposed. The active site architecture shows clear evidence of having arisen by convergent evolution.

Serial femtosecond crystallography: A revolution in structural biology

“…From top left: photosystem I (3PCQ 2 ), FABP3 (3WXQ 3 ), purple bacterial reaction center (4CAS 4 ), lysozyme (4ET8 5 ), cathepsin B (4HWY 6 ), 5-HT2B (4NC3 8 ), photosystem II 1 ), δ-opiod receptor (4RWD 10 ), smoothened receptor ( 11 ), Cry3A (4QX0 14 ), CPV17 polyhedron (4S1K 15 ), diacylglycerol kinase (4UYO 17 ), xylose isomerase (4W4Q 3 ), photoactive yellow protein (4WL9 20 ), SR Ca 2+ -ATPase (4XOU 22 ), angiotensin II receptor (4YAY 24 ), phycocyanin (4Z8K 25 ), luciferin-regenerating enzyme (5D9B 27 ), C-phycocyanin (4ZIZ 29 ), rhodopsin-arrestin complex (4ZWJ 30 )…”

A Bright Future for Serial Femtosecond Crystallography with XFELs

“…One of the most successful applications of SFX has been the structural study of membrane proteins using LCP as the crystallization and crystal delivery medium, which led to structure determination of several GPCRs [13–19] including the signaling complex between GPCR and arrestin [20], the membrane enzyme diacylglycerol kinase [102], and the light-activated proton pump bacteriorhodopsin [103]. …”

Structural basis for catalysis at the membrane-water interface

“…The structure of DgkA from Escherichia coli ( Ec DgkA) , was solved without and with bound adenylylmethylenediphosphonate (ACP; non-hydrolyzable ATP analogue) to 2.05 Å and 2.7 Å resolution respectively by x-ray crystallography from crystals grown in LCP [21, 22]. Both structures contained bound monoacylglycerol (MAG) lipids of different lengths from the LCP formulation (7.8 MAG in the apo structure; 9.9 MAG in the ACP bound structure).…”