NMR Spectroscopy of Intrinsically Disordered Protein Complexes

The main goal of the proposed research project is to establish a NMR methodological framework to elucidate the atomistic details of protein/protein and protein/ligand recognition involving intrinsically disordered proteins (IDPs). Specifically, we plan to study, with atomic resolution, changes in the structural dynamics of IDPs upon binding to membrane systems, small molecules and cognate proteins binding partners as well as the impact of post- translational modifications (long-chain acylation and phosphorylation) on the structural dynamics of IDPs. The two IDPs under investigation are GAP-43 and BASP1, two neuronal proteins involved in neuronal plasticity and synaptic vesicle fusion. IDPs have attracted a lot of attention over the last decade due to both their fascinating structural properties and their involvement in important physiological and pathological processes. These proteins challenge the old structure-function paradigm; not only are they lacking stably folded tertiary structures, but their intrinsic flexibility actually has a significant impact on their biological functionality. The sampling of a vast and heterogeneous conformational space endows IDPs with enormous potential to interact with, and control, multiple binding partners at once. Since IDPs don`t follow the old structure-function paradigm, the way they interact with their partners remains elusive and is a very active field of research. Although, several models describing molecular interaction involving IDPs have been proposed (folding upon binding, fuzzy complex) none of these models satisfyingly explain the molecular mechanisms relevant for the ability of IDPs to interact with diverse partners (small molecules, ions, proteins) sequentially or all at once. The intended project aims at filling this gap. The two proteins of interest (Growth Associated Protein 43 (GAP-43) and the Brain Acid Soluble Protein 1, BASP1) are functionally related as they are both found in neurons where they sequester phosphatidylinositol-4,5- bisphosphate (PIP2) in lipid rafts. They are both undergoing long-chain acylation. Both bind Ca2+, are the substrate of Protein Kinase C (PKC) and interact with calmodulin (although by very distinct modes). Surprisingly, although they share so similar functionalities, GAP-43 and BASP1 share no significant sequence identity. Therefore these two proteins are models of choice in order to understand how different (but functionally related) IDPs exploit their various conformational ensembles for accommodation of their respective partners. Additionally, BASP1 also acts as a tumor suppressor; therefore our results might be of relevance in cancer biology.

The main goal of the proposed research project was to establish a NMR methodological framework to elucidate the atomistic details of protein recognition involving intrinsically disordered proteins (IDPs). Although their enormous relevance for biology and biomedical research is well-established detailed knowledge about the underlying molecular mechanisms is still missing. In the successfully completed project we studied, with atomic resolution, changes in the structural dynamics of IDPs upon binding to membrane systems, small molecules and cognate proteins binding partners as well as the impact of post-translational modifications on the structural dynamics of IDPs. To this end novel techniques were developed to prepare specifically modified proteins and visualize their complex structural dynamics. These novel techniques provided unprecedented insight into the sampling of the vast and heterogeneous conformational space of IDPs and an explanation how they interact with, and control, multiple binding partners at once, and will support the development of novel therapeutic strategies to combat cancer or neurodegenerative diseases.