Mechanistic Insights into the Specificity of Glycosaminoglycan Interactions with Regulatory Proteins
Final Report Abstract
The project aimed to quantitatively investigate the specificity of the interaction between glycosaminoglycans (GAG) and regulatory proteins. GAGs are long-chain linear sugars that are characterized by a high degree of sulfatation, leading to high charge density. In particular, the question was to what extent the recognition of GAG by proteins is realized by nonspecific electrostatics or by a pattern of specific interactions, such as hydrogen bonds. First, the interaction of the small cytokine interleukin-8 (IL-8) with a series of negatively charged molecules was investigated. In our preliminary work, we had investigated the interaction of various GAGs with IL-8 in detail and created structural models. The aim here was to clarify whether peptides with a similar charge density and distribution to GAGs also bind the same epitopes as GAGs. For this purpose, a library of peptides was synthesized that differed in their charge density. The binding of these peptides to IL-8 was primarily investigated using nuclear magnetic resonance spectroscopy techniques. In addition, computer-based docking studies and molecular dynamics simulations were carried out. Structural models of the interaction of the peptides with IL-8 were created. It was shown that the peptides indeed bind the same polybasic clusters on the protein. It was shown that the peptides actually bind the same polybasic clusters to the protein. However, the affinity of the peptides for IL-8 is orders of magnitude lower, which can be attributed to entropic effects. In contrast to GAG, the unbound peptides are present in a random and highly dynamic structure; upon binding to IL-8, the conformational space of the peptides is very significantly reduced, which is associated with high entropy loss. We also investigated the interaction of novel dendritic GAGs with IL-8, which are of interest for potential regenerative processes. In the course of the project, a method was published in which soluble paramagnetic probes were used to measure the surface charge of proteins. Since the interaction of GAG with regulatory proteins is very important for its surface charge, we investigated whether the method is suitable for describing the binding of small molecules to proteins. It turned out that this is indeed a possibility to experimentally describe the interaction of proteins with different ligands and computer simulations confirmed the experimental results. In the second part of the project, the interaction of cathepsins with GAG will be investigated quantitatively using both methods. Cathepsin B has a very positive surface charge, so that an unspecific interaction with negatively charged molecules is to be expected. We were able to successfully establish the expression and function of catepsin B, but the biochemical work took much longer than originally assumed. Initial NMR studies showed that some catepsin B signals are shifted by GAG binding. However, as no assignment of the NMR signals was available, we were unable to carry out any further structural investigations. For a possible continuation of the project, cathepsin B would have to be produced with isotope labeling (2H, 13C, 15N) and assigned using triple resonance methods. Furthermore, crystallographic work provided the structure of the catepsin B complex with heparin with a resolution of 1.8 Å. All experimental work was carried out in our working group, while the computer-based techniques were performed by our cooperation partner Dr. Sergey Samsonov in Poland (University of Gdańsk).
Publications
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"Untersuchungen zur Interaktion von Glykosaminoglykanen mit löslichen regulatorischen Proteinen“ Dr. rer. nat., Fakultät für Lebenswissenschaften, 2023
Christian Schulze
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Ligand binding of interleukin-8: a comparison of glycosaminoglycans and acidic peptides. Physical Chemistry Chemical Physics, 25(36), 24930-24947.
Schulze, Christian; Danielsson, Annemarie; Liwo, Adam; Huster, Daniel; Samsonov, Sergey A. & Penk, Anja
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Detecting Protein‐Ligand Interactions with Nitroxide Based Paramagnetic Cosolutes. Chemistry – A European Journal, 30(18).
Penk, Anja; Danielsson, Annemarie; Gaardløs, Margrethe; Montag, Cindy; Schöler, Andrea; Huster, Daniel; Samsonov, Sergey A. & Künze, Georg
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Dimerization and Crowding in the Binding of Interleukin 8 to Dendritic Glycosaminoglycans as Artificial Proteoglycans. Chemistry – A European Journal, 30(13).
Dürig, Jan‐Niklas; Schulze, Christian; Bosse, Mathias; Penk, Anja; Huster, Daniel; Keller, Sandro & Rademann, Jörg
