Structural investigation of the human Guanylate Binding Protein 1 in solution

Lorenz, Charlotte; Richtering, Walter (Thesis advisor); Herrmann, Christian (Thesis advisor)

Aachen (2019)
Dissertation / PhD Thesis

Dissertation, RWTH Aachen University, 2019


Guanylate-binding proteins are key players of the immune response of organisms and therefore essential for survival. The human Guanylate Binding Protein 1 (hGBP1) is an important representative of this family that was discovered very early due to its high affinity to guanosine-5’-triphosphate, the -diphosphate as well as to the monophosphate. Due to the heterologous expression in E.coli cells for protein purification, the protein was purified for many years without posttranslational modifications, though it was shown already in 1996 that hGBP1 is modified in vivo with a farnesyl moiety attached. With the unmodified protein, the crystal structure of the full-length protein was solved in 2000, and in 2006 as dimers of a truncated version consisting of only the GTPase binding domains. As nucleotide-activated oligomerization was observed for other family members already, hGBP1’s oligomerization behaviour was studied as well leading to a proposed hydrolysis cycle of an initial inactive monomer, which dimerizes after nucleotide addition and tetramerizes in the hydrolysis transition state. In contrast to these in vitro studies, large supramolecular clusters were observed in vivo that are exclusively forming after posttranslational farnesylation of hGBP1.In the course of this PhD thesis, the fundamental differences between unmodified and farnesylated hGBP1 have been studied. Part I mainly focusses on the structural flexibility and dynamics of the monomeric unmodified protein occuring in the absence of nucleotides, but also in presence of nucleotides, that are leading to the later observed large structural rearrangements necessary for dimerization of a second C-terminal interaction side next to the earlier described GTPase binding domain. Consecutively, the next part II focusses on the effect of the farnesyl moiety, which prevents those structural rearrangements as observed in part I probably by locking the hydrophobic C-terminus to a hydrophobic patch that is located close to the first interaction side. Thereby, the earlier observed dynamics of the monomer are only possible after nucleotide addition, allowing thereby a very fine tuned and specific signal transduction in cells by protein expression and nucleotide binding. The last part III focusses on farn-hGBP1 oligomerization occuring after nucleotide addition. In contrast to the earlier described dimers and tetramers proposed for unmodified hGBP1, large oligomeric clusters are observed for farn-hGBP1 after activation. Different techniques are used to obtain real space information like cryo transmission electron microscopy and fluorescence microscopy, probing aggregation properties on different length scales. Additionally, time resolved reciprocal space information are gained from scattering techniques like small angle X-ray and light scattering. The combination of all methods allows to describe the polymerization process to take place mediated by nucleating cores, so-called discs, to large-scaled protein aggregates after a while. This work can thereby contribute to describe the in vivo observed effects of phase separation and appearance of ‘vesicle-like structures’ of farn-hGBP1 after activation by nucleotides.