Low-complexity domain protein molecular structure, conformational dynamics, and inter-protein interactions in human health and disease

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Project Summary/Abstract The dynamic assembly of biomolecules within a living cell is vital for the spatial and temporal organization of biological function. In forming RNA granule membraneless organelles and intermediate filaments in the cell cytoskeleton, cells leverage the self-assembly properties of protein sequences with reduced amino acid diversity. These low complexity protein domains have only recently come to light as essential players in these processes. Thirty percent of the proteins coded by the human genome contain a domain of this type, highlighting the central importance of these sequences for life. In humans, pathogenic genetic mutations and altered expression levels, in addition to functional post-translational modifications and protein-protein interactions, modulate the assembly processes of these proteins. Linked by the common involvement of low complexity domain proteins, this proposal outlines two lines of research focused on a fundamental mechanistic understanding of how the proteins that compose RNA granules and intermediate filament networks assemble to achieve the macroscopic behavior observed in living cells. A multifaceted biophysical approach employing cutting-edge nuclear magnetic resonance and cryo-electron microscopy will allow characterization of the molecular structure and conformational dynamics of these proteins in biologically relevant assemblies. These biophysical studies will be coupled with other spectroscopies, biochemical assays, and protein engineering to form more comprehensive models of how low complexity domain proteins assemble temporally and spatially. The results of these efforts will provide a mechanistic description of how these assembly processes and their associated control mechanisms are modulated by point mutations and altered protein expression levels linked to motor neuron disease, dementia, muscular dystrophy, and cancer. The in vitro work proposed here will provide detailed and testable models regarding the function of in vivo biological assemblies involved in RNA metabolism and the cell cytoskeleton. In the broader context of human health, the molecular characterizations of disease-relevant low complexity domain proteins and their interacting molecular partners will provide a base of knowledge useful for the exploration of these systems as clinical biomarkers and will also facilitate the development of antibody and small molecule therapeutics. Beyond the specific biological systems discussed in this proposal, the tools and methodologies employed here are expected to have applicability and impact on investigations of the thirty percent of the proteins in the human genome that contain a low complexity domain.