Although the interaction between interleukin-8 (IL-8) and glycosaminoglycans (GAGs) is crucial for the mediation of inflammatory effects, little is known about the site specificity of this interaction. Therefore, we studied complexes of IL-8 and heparin (HEP) as well as other GAGs in a multidisciplinary approach, involving site-directed mutagenesis, mass spectrometry, fluorescence and solution NMR spectroscopy as well as computer modeling. The interaction between GAG and IL-8 is largely driven by the amine groups of the lysine and the guanidinium groups of arginine side chains. However, due to fast exchange with the solvent, it is typically not possible to detect NMR signals of those groups. Here, we applied reductive (13)C-methylation of the lysine side chains providing sensitive NMR probes for monitoring directly the sites of GAG interaction in (1)H-(13)C correlation experiments. We focused on the lysine side chains K25, K28, K59, K69 and K72 of IL-8 (1-77), which were reported to be involved in the binding to GAGs. The NMR signals of these residues were assigned in (1)H-(13)C HSQC spectra through the help of site-directed mutagenesis. NMR and fluorescence titration experiments in combination with molecular docking and molecular dynamics simulations were applied to investigate the involvement of each lysine in the binding with HEP and various GAG hexasaccharides. We identified K25, K69 and K72 to be the most relevant binding anchors of IL-8(1-77) for the analyzed GAGs.

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. Furthermore, functionally relevant interprotomer interactions are identified by DNP-enhanced MAS NMR in combination with site-directed mutagenesis and functional assays.