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Strength in Numbers: How Multivalent WW-PPXY Interactions Regulate Cell Signaling Público Deposited

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  • Cell signaling is often mediated by protein-protein interactions, which must be specific, tunable, and transient to allow agile responsiveness to cellular messages. Due to their unique properties, multivalent, intrinsically disordered proteins make ideal candidates to accomplish these vital tasks. A single protein with multiple binding sites may bind numerous partners, leading to diverse but integrated cellular outcomes. However, despite their high prevalence and importance, several challenges have hindered the detailed characterization of multivalent, disordered proteins and complexes. First, these proteins are often low yielding and have poor stability and solubility. Second, the inherent dynamic nature of multivalent, disordered proteins often translates to the formation of intricate heterogenous mixtures of protein complexes. Third, few biophysical techniques are amenable to studying these complicated and dynamic systems. In this work, we overcome these challenges to elucidate the mechanisms of complex assembly for three sets of interactions. The defining feature of each interaction is multivalency, as each protein contains multiple recognition sites. At the heart of these interactions is the multivalent scaffolding protein, Angiomotin-Like 1 (AMOTL1), which plays essential roles in cell growth regulation, cell polarity, shape, and tight junction formation. Important for this thesis is a largely disordered segment of AMOTL1 with three short linear motifs of the sequence L/PPXY (L=leucine, P=proline, Y=tyrosine, and X=any amino acid, hereafter referred to as PPXY). The partner proteins – YES-associated protein (YAP), Kidney and Brain-expressed protein (KIBRA), and neural precursor cell-expressed developmentally downregulated 4 (NEDD4) – contain multiple repeats of the globular WW domain, an arrangement that facilitates multivalent interactions with AMOTL1 that are functionally relevant. Thus, understanding how each protein complex assembles in the context of multivalency and how AMOTL1 discriminates between its partners could inform critical cellular processes. Chapter 1 introduces unique features that make intrinsically disordered proteins ideal candidates in multivalent interactions. Emphasis is placed on specific multivalent interactions mediated by the largely disordered PPXY-motif region of AMOTL1 and its multivalent globular WW domain-containing partners. In the following chapters, we provide in-depth molecular biophysics studies of the solution properties of the disordered PPXY-rich segment of AMOTL1 and its interaction with YAP (Chapter 2), KIBRA (Chapter 3) and NEDD4 (chapter 5). Unique characteristics of each interaction are highlighted. Chapter 4 is a study of the solution properties of the multiple WW domains of NEDD4, with emphasis on structural dynamics and global communication between the domains. Chapter 6 summarizes key findings, impact, and future directions of this work.
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