Resolving the functional mechanism of the lipid regulator diacylglycerolkinase by solid-state NMR
Final Report Abstract
Escherichia coli diacylglycerol kinase (DGK) is an integral membrane protein, which catalyses the ATP-dependent phosphorylation of diacylglycerol (DAG) to phosphatic acid (PA). It is a unique trimeric enzyme, which does not share sequence homology with typical kinases. It exhibits a notable complexity in structure and function despite of its small size. Here, chemical shift assignment of wild-type DGK within lipid bilayers was carried out based on 3D MAS NMR, utilizing manual and automatic analysis protocols. Upon nucleotide binding, extensive chemical shift perturbations could be observed. These data provide evidence for a symmetric DGK trimer with all of its three active sites concurrently occupied. Additionally, we could detect that the nucleotide substrate induces a substantial conformational change, most likely directing DGK into its catalytic active form. Functionally relevant intra- and interprotomer contacts are identified by DNP-enhanced MAS NMR in combination with site-directed mutagenesis and functional assays.
Publications
- Conformation and topology of diacylglycerol kinase in E.coli membranes revealed by solid-state NMR spectroscopy. Angew. Chem. Int. Ed. Engl. 2014, 53 (22), 5624-8
Chen, Y.; Zhang, Z.; Tang, X.; Li, J.; Glaubitz, C.; Yang, J.
(See online at https://doi.org/10.1002/anie.201311203) - Overcoming Volume Selectivity of Dipolar Recoupling in Biological Solid-State NMR Spectroscopy. Angew. Chem. Int. Ed. Engl. 2018
Tosner, Z.; Sarkar, R.; Becker-Baldus, J.; Glaubitz, C.; Wegner, S.; Engelke, F.; Glaser, S. J.; Reif, B.
(See online at https://doi.org/10.1002/anie.201805002) - Global response of diacylglycerol kinase towards substrate binding observed by 2D and 3D MAS NMR. Scientific Reports 2019
Möbius, K.; Kazemi, S.; Güntert, P.; Jakob, A.; Heckel, A.; Becker-Baldus, J.; Glaubitz, C.
(See online at https://doi.org/10.1038/s41598-019-40264-8)
