Structural Analysis of Receptor Interactions and Structure-based Protein Engineering of Prenyltransferases

Abstract

This thesis combines three unrelated projects with diverse biological background. For all projects, the scientific questions were addressed by X-ray structure determination of the target molecules. Since 2019, SARS-CoV-2 causes an ongoing pandemic with 460 million infections and 6 million deaths worldwide (date 15.03.2022) and up to now, all available vaccines offer no full, long-life protection against an infection with this virus. Nbs against the receptor binding domain (RBD) of SARS-CoV-2 were produced and the affinity to the RBD as well as the virus neutralisation capabilities were determined. In this work, two high-affinity binders, NM1226 and NM1230, were structurally investigated in complex with the RBD to analyse the binding properties on an atomic level. Both Nbs bind to the RBD with a non-overlapping interface. As a consequence, both Nbs utilise different strategies to prevent RBD interactions and therefore can block binding to the host cell entry receptor angiotensin converting enzyme 2 (ACE2). By combining these two Nbs, a new biparatopic nanobody (biNb) NM1267, which binds to the RBD with picomolar affinity, was created by our cooperation partner. Additionally, analysis of the binding ability to emerging SARS-CoV-2 variants revealed robust binding of the biNb to the RBD. An assay was established with the biNb to analyse the antibody composition of SARS-CoV-2 infected patients that allows to estimate the amount of neutralising antibodies in patients. Secondary metabolites are chemically diverse, small molecules featuring antifungal, an- tibacterial or anti-inflammatory properties. Some of these compounds possess a prenyl entity, which often enhances their bioactivity. This modification is carried out by prenyl- transferases in a chemo- and regioselectivity reaction. The selectivity of the enzymatic prenylation offers a substantial advantage compared to de novo synthesis, which makes these enzymes valuable targets for biotechnological applications. Over the last ten years, a subgroup of prenyltransferases, the dimethylallyl tryptophan synthases (DMATS) were analysed in detail with the aim of controlling the reaction mechanism and influencing the substrate specificity. The work presented here improved the general understanding of the enzyme family and provides the first 5-DMATS structure in complex with its natural substrates l-tryptophan and dimethylallyl pyrophosphate (DMAPP). In addition, a ligand- bound structure of a C-6 prenylating DMATS (6-DMATS) was obtained. By investigation of the structures, crucial residues for catalysis were determined in both enzymes. The regioselectivity of 6-DMATS could be switched towards a 5-DMATS by structure-based engineering of the enzyme. This principle could be applied to other prenyltransferases to specifically modify the enzymatic efficiency for biotechnological production of new compounds.

Sialic acid-binding immunoglobulin-like lectins (Siglecs) are receptors that are mainly present on haematopoietic cells and recognise sialic acid residues on glycoproteins. Siglec-11 is expressed on macrophages and microglia and has, upon binding to α2,8-linked polysialic acid (polySia), an inhibitory regulatory role on immune cell activation. PolySia is found as rare decoration on glycoproteins mainly expressed during development, and in plastic regions of the brain of healthy adults. In addition, some pathogens, such as Escherichia coli serotype K1, and tumours present polySia on their surface to exploit the inhibitory effect on immune cell activation and evade the immune system. Compared to other Siglecs, whose specificity is mainly determined by the linkage and modification of the last few entities of the ligand glycan, Siglec-11 interaction with polySia differs in fundamental aspects. Only polySia with a degree of polymerisation larger than 20 has an anti-inflammatory effect on human macrophages. Therefore, other structurally characterised interactions of Siglecs with ligands offer no sufficient model to explain polySia engagement by Siglec-11. Siglec-11 is characterised by a N-terminal ligand binding V-set and three Ig-like C2-set domains. In this work, Siglec-11 variants with a varying number of extracellular domains were expressed and purified for structural studies and affinity determination to polySia. A structure of Siglec-11 V-set domain could be obtained at 2.15 Å resolution and lays the foundation for further structural studies with polySia.