Abstract
Diacylglycerol kinase (DgkA) is a small integral membrane protein, responsible for the ATP-dependent phosphorylation of diacylglycerol to phosphatidic acid. Its structures reported in previous studies, determined in detergent micelles by solution NMR and in monoolein cubic phase by X-ray crystallography, differ significantly. These differences point to the need to validate these detergent-based structures in phospholipid bilayers. Here, we present a well-defined homo-trimeric structure of DgkA in phospholipid bilayers determined by magic angle spinning solid-state NMR (ssNMR) spectroscopy, using an approach combining intra-, inter-molecular paramagnetic relaxation enhancement (PRE)-derived distance restraints and CS-Rosetta calculations. The DgkA structure determined in lipid bilayers is different from the solution NMR structure. In addition, although ssNMR structure of DgkA shows a global folding similar to that determined by X-ray, these two structures differ in monomeric symmetry and dynamics. A comparative analysis of DgkA structures determined in three different detergent/lipid environments provides a meaningful demonstration of the influence of membrane mimetic environments on the structure and dynamics of membrane proteins.
Highlights
Diacylglycerol kinase (DgkA) is a small integral membrane protein, responsible for the ATPdependent phosphorylation of diacylglycerol to phosphatidic acid
Magic angle spinning (MAS) solid-state NMR spectroscopy has emerged as a powerful technique for structure determination of membrane proteins (MPs) in phospholipid bilayers[18,19,20,21], which are closer to physiological conditions compared to other membrane mimetic environments
SsNMR spectra of DgkA display considerable conformational heterogeneity as indicated by the 13C linewidth of 150–200 Hz in the spectra of the U-13C, 15N-DgkA sample, NMR resonances for 78% of the residues in no-cys-DgkA mutant were assigned based on a set of triple resonance sequential dipolar-coupling-based 3D solid-state NMR (ssNMR) spectra (BMRB ID: 50508)
Summary
Diacylglycerol kinase (DgkA) is a small integral membrane protein, responsible for the ATPdependent phosphorylation of diacylglycerol to phosphatidic acid. Its structures reported in previous studies, determined in detergent micelles by solution NMR and in monoolein cubic phase by X-ray crystallography, differ significantly. These differences point to the need to validate these detergent-based structures in phospholipid bilayers. A comparative analysis of DgkA structures determined in three different detergent/lipid environments provides a meaningful demonstration of the influence of membrane mimetic environments on the structure and dynamics of membrane proteins. The structure from solution NMR has a domain-swapping property while the one from X-ray crystallography has not These differences suggest a possible perturbation of DgkA structure by the detergent environments and highlight the need for the validation of these distinct structures in native-like lipid bilayers. Comparative analysis of DgkA structures resolved in three different detergent/lipid environments using three structural methodologies provides valuable insights into the influence of membrane mimetic environments on the structure of a multispan α-helical MP
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