Hub proteins bind a large number of partners to facilitate structural changes and downstream protein interactions. LC8, a highly conserved protein homodimer, is a unique hub that regulates the activity of proteins in a wide range of cellular processes by binding to intrinsically disordered regions. With many of these systems, LC8 is known to function as a dimerization engine, bringing together two disordered chains to facilitate structural change. But for the significant and increasingly large class of multivalent LC8 binding partners, the role of LC8 binding is less clear.
This thesis reports on work aimed at analyzing the structure and function of complexes formed between LC8 and three multivalent protein partners: ASCIZ, Chica, and Nucleoporin 159. Four chapters of original work include one review, two primary research reports, and one unpublished study. The review (chapter 2) classifies different types of intrinsically disordered multivalent protein assemblies and highlights the unique features of complexes formed between LC8 and its multivalent protein partners. Chapter 3 is an in-depth study of the factors that influence partner protein binding to LC8 using the multivalent protein Chica as a model system. The ‘anchored flexibility model’ of LC8 motif recognition is introduced, which proposes that a few residues are essential for binding, while others modulate binding affinity. This model and the structural data presented in this work enhance our understanding of how LC8 recognizes and binds to its protein partners. Chapter 4 focuses on the structural and functional characterization of the protein ASCIZ, a transcription factor for LC8. This work demonstrates how the concerted action of multiple binding sites in an intrinsically disordered region enable a gradient of ASCIZ activity and finely tune the transcriptional level of LC8. Chapter 5 is a biophysical examination of the structure and assembly process of the complex formed between Nucleoporin 159 and LC8. Finally, chapter 6 discusses the impacts and highlights of the reported work, and compares the properties of the multivalent protein complexes presented in chapters 3-5. Individually, these results provide a detailed biophysical description of three unique macromolecular protein assemblies. Together, they emphasize the structural and functional flexibility of LC8 as hub protein.