Graduate Thesis Or Dissertation
 

Specificity, Allostery, and Multivalency in Binding to the Hub Protein LC8

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  • Interactions between proteins are essential to life, driving and regulating a majority of processes within all living cells. Study of protein-protein interactions reveals that some proteins act as hubs within networks of interactions, binding to many partner proteins. These hubs therefore are of particular importance to understanding protein function, interwoven as they are with dozens of biological functions. LC8 is one such hub protein, binding to over 100 known clients and playing a role in many unrelated pathways. LC8 binding, mediated by a short linear motif in client proteins, induces a dimeric structure on clients, leading the protein to be referred to as a dimerization engine. This thesis discusses the function of LC8, examining both the general properties of LC8 that facilitate LC8-client binding, and documenting and characterizing new LC8-binding proteins. Each of the three chapters of original work is a report of primary research. Chapter 2 is a detailed investigation of the thermodynamics of LC8 binding, which necessitated the development of a new method of analysis built on principles of Bayesian statistics. This method allowed us to measure detailed thermodynamics of LC8 binding, and demonstrate that LC8 favors a fully bound state, consistent with its function as an engine for dimerization. Chapter 3 is concerned with characterizing the LC8-binding linear motif, and development of a tool for prediction of LC8 binding. We collate a database of LC8-binding proteins and find that residues flanking the core motif sequence play an important role in regulating binding. The predictive tool uses a library of known LC8-binding and non-binding sequences to generate a scoring matrix for potential clients and has already been adopted by researchers studying LC8 interactions. In chapter 4 we present a characterization of a new LC8-binding protein named Kank1. Kank1, a cytoskeletal regulator found at the cell cortex, binds LC8 multivalently, forming a large complex consisting of at least five LC8 dimer units. The complex forms with significant cooperativity, and unlike many multivalent LC8-interacting proteins, forms a homogenous stable oligomer, indicating that the complex may play a structural role, rigidifying the scaffold of Kank1. Lastly, chapter 5 discusses the impact of this work, and highlights of the work presented in each chapter. It additionally presents ongoing and future steps in the study of LC8 interactions. This thesis additionally contains two appendices reporting primary research that is unrelated to LC8. The first is concerned with a protein from the Peroxiredoxin family of redox proteins. Peroxiredoxins unfold during catalysis, and we demonstrated that our model peroxiredoxin unfolds transiently in absence of catalysis, emphasizing that the protein is finely structurally tuned for catalysis. The second appendix discusses the nucleocapsid of the SAR-CoV-2 virus, examining the protein’s interaction with RNA, which is essential to viral replication. We find that the protein can interact both specifically and nonspecifically with RNA, and that nonspecific binding is correlated to liquid-liquid phase separation, which is believed to be essential to some viral functions.
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