Reinier Damman

Cells rely on their ability to sense the environment in order to quickly and adequately respond to an ever changing environment. They are able to respond by means of proteins, which are the molecular machines of cells and allow for a proper cellular homeostasis. The relationship between the structure and function of proteins is complex but of crucial importance for cellular homeostasis. Activation of proteins frequently goes hand-in-hand with conformational changes as a consequence of ligand binding. Previous publications suggested that these conformational changes occur due to intrinsically dynamic protein domains. Often the physiological environment also imposes a significant effect on their functioning. When new proteins are synthesised, they are post-translationally modified in eukaryotic cells which can be important for their function. Moreover, membrane proteins are often embedded in specialized lipid environments that directly interact with the protein. Therefore, it is of great importance to study the structure of proteins under physiologically relevant conditions. This actually imposes a problem on structural biology techniques as they generally require a simplified and isolated system to obtain high-resolution data. In this sense, solid-state Nuclear Magnetic Resonance (ssNMR) spectroscopy is unique as it is not limited by the sample size and complexity. Additionally, ssNMR can provide insights into the dynamic- and rigid profile of proteins simultaneously. In this thesis, we describe studies where we studied the dynamics, activation and functioning proteins in a complex or native lipid environment using ssNMR and molecular biology techniques. These systems play a central role in the development of cancer, e.g. breast cancer, but are also involved in neurodegenerative diseases. In recent years there has been an increase in interest towards the development of novel ssNMR methods which opens up the possibility to measure cellular- and even in-cell-ssNMR samples.

In chapter 2, we present an overview of recent literature describing the development for prokaryote- and eukaryote samples. These methods offer the possibility to specifically label a single protein by enriching it with NMR-active isotopes inside a cellular system or they relate to approaches to deliver a NMR-visible protein inside an unlabelled cell. The literature overview emphasises the increased interest in this field and underscores the relevance of performing structural studies inside a native environment.

Previous work performed by Kaplan and colleagues highlighted the importance of the native environment on the intrinsic dynamic character of the extracellular domain (ECD) of the Epidermal Growth Factor Receptor (EGFR), but a role for this dynamics has never been described. Chapter 3 presents new data, in which we made use of allosteric nanobodies showing that conformational freedom is essential in the functioning of EGFR, because the nanobody locks an important hinge region of the receptor. By making point mutations in the nanobody, we were able to increase the dynamic behaviour of the ECD which lead to increased activity and internalisation rates of EGFR. Because the dynamics of the ECD is crucial in the activation of EGFR, we wondered whether this is a general feature of growth factor receptors. In chapter 4, we adapted the method previously used for the EGFR studies, for a cell line which has an endogenous overexpression of HER2, a family member of the receptor tyrosine kinase family. HER2 is a unique receptor as until date, no activating ligand has been found. This implies that receptor activation is dependent on heterodimerisation. Here we present ssNMR measurements that, in contrast to the general assumption, suggest that the ECD of unstimulated HER2 is dynamic. EGF-dependent stimulation of HER2 results in an increase in phosphorylation levels and increased ECD rigidity. Finally, we made use of an anti-EGFR nanobody, described in chapter 3, which induces a small rigidification of the EGFR ECD by locking a receptor hinge region. This nanobody was shown to indirectly increase phosphorylation levels of HER2 and also stabilize the HER2 ECD. We conclude that the presence of a rigid dockingpartner, and therefore not ligand binding, is the driving force for HER2 phosphorylation and oligomerization.

Chapter 5 describes a different system where the dynamic character is important in protein functioning. We made use of a combination of solution- and solid-state NMR to derive an atomistic model on phase separation. Our data shows that phase separation coincides with increased rigidity of a mobile unstructured region. We do not observed significant conformational changes. The intermolecular transient and weak interactions of the unstructured regions with the dimerization domain allow the formation of the dynamic macromolecular structure. Even though structural research is being performed in situ when possible, there is still a gap between the environment of cells within a human body and cells that are being cultured in a laboratory setting. Therefore, researchers in biology have developed 3-dimensional cell culture techniques which attempt to mimic the cellular environment inside a body. In chapter 6, we have developed a large-scale method which results in the formation of homogenous spheroids, which function as tumour model systems. We subjected them to ssNMR experiments in order to record structural data directly on the spheroids. Moderate MAS speeds did not disrupt the spheroid which enables structural studies directly on these model systems.

In conclusion, this thesis presents work which confirms the importance of the dynamic character of proteins and thereby links the structure and function of proteins. Additionally, it is important to perform such studies within the native environment. The combination of ssNMR and cell biological experiments was central in studying the structure-function relationship.