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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 ?-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.

SUBMITTER: Li D

PROVIDER: S-EPMC4703834 | biostudies-literature | 2015 Dec

REPOSITORIES: biostudies-literature

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Ternary structure reveals mechanism of a membrane diacylglycerol kinase.

Li Dianfan D Stansfeld Phillip J PJ Sansom Mark S P MSP Keogh Aaron A Vogeley Lutz L Howe Nicole N Lyons Joseph A JA Aragao David D Fromme Petra P Fromme Raimund R Basu Shibom S Grotjohann Ingo I Kupitz Christopher C Rendek Kimberley K Weierstall Uwe U Zatsepin Nadia A NA Cherezov Vadim V Liu Wei W Bandaru Sateesh S English Niall J NJ Gati Cornelius C Barty Anton A Yefanov Oleksandr O Chapman Henry N HN Diederichs Kay K Messerschmidt Marc M Boutet Sébastien S Williams Garth J GJ Marvin Seibert M M Caffrey Martin M

Nature communications 20151217

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 ...[more]

PMID: 26673816

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Project description:The validation of protein structures through functional assays has been the norm for many years. Functional assays perform this validation for water-soluble proteins very well, but they need to be performed in the same environment as that used for the structural analysis. This is difficult for membrane proteins that are often structurally characterized in detergent environments, although functional assays for these proteins are most frequently performed in lipid bilayers. Because the structure of membrane proteins is known to be sensitive to the membrane mimetic environment, such functional assays are appropriate for validating the protein construct, but not the membrane protein structure. Here, we compare oriented sample solid-state NMR spectral data of diacylglycerol kinase previously published with predictions of such data from recent structures of this protein. A solution NMR structure of diacylglycerol kinase has been obtained in detergent micelles and three crystal structures have been obtained in a monoolein cubic phase. All of the structures are trimeric with each monomer having three transmembrane and one amphipathic helices. However, the solution NMR structure shows typical perturbations induced by a micelle environment that is reflected in the predicted solid-state NMR resonances from the structural coordinates. The crystal structures show few such perturbations, especially for the wild-type structure and especially for the monomers that do not have significant crystal contacts. For these monomers the predicted and observed data are nearly identical. The thermostabilized constructs do show more perturbations, especially the A41C mutation that introduces a hydrophilic residue into what would be the middle of the lipid bilayer inducing additional hydrogen bonding between trimers. These results demonstrate a general technique for validating membrane protein structures with minimal data obtained from membrane proteins in liquid crystalline lipid bilayers by oriented sample solid-state NMR.

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Ternary structure reveals mechanism of a membrane diacylglycerol kinase.

Publications

Ternary structure reveals mechanism of a membrane diacylglycerol kinase.

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