Investigating Protein Complex Dynamics: Analysis of Cullin-Ring Ligase Machinery through Development of Quantitative Cross-linking Mass Spectrometry Strategies
Protein assemblies represent the workhorses of the cell, forming the basis of all cellular processes. Their biological roles are intimately associated with their topologies, making the structural elucidation of proteins and protein complexes a critical requirement to understanding their function. While traditional structural biology approaches have greatly contributed to our current understanding of protein structure, they are ill-suited for analyzing the conformational dynamics associated with heterogenous protein complexes and their protein-protein interactions (PPIs). As a result, there is a growing demand for the development of new structural approaches to elucidate the impact of protein dynamics on the regulation of integral biological processes required for cell homeostasis. In particular, cross-linking mass spectrometry (XL-MS) has arisen in recent years as a popular hybrid structural technique for the topological determination of conformationally and compositionally heterogenous protein complexes. However, most studies utilizing cross-linking thus far have been limited to the determination of static protein structures.
Here, I focus on the development and application of quantitative cross-linking mass spectrometry (QXL-MS) strategies to determine how conformational dynamics of cullin-RING ligases (CRLs) dictate and regulate their ubiquitinating activity. Proteasomal dysregulation has been associated with a wide range of human pathologies, from diabetes and various forms of cancer to autoimmune and neurodegenerative disorders. As a result, the PPIs associated with CRL assemblies represent potential targets for therapeutic intervention. A comprehensive understanding of E3 ligase structure and conformational dynamics is critical for the development of pharmacological drugs that selectively inhibit or upregulate their function. However, such heterogenous assemblies are notoriously difficult to study using traditional structural biology techniques.
While this thesis focuses on the application of quantitative XL-MS strategies to study cullin-RING ligase machinery, these platforms represent versatile methodologies that can be universally employed for structural studies on a wide range of protein systems.