Phafin2, an adaptor protein, is involved in various cellular processes, such as apoptosis, autophagy, endosomal cargo transportation, and macropinocytosis. Two domains, namely, PH and FYVE, contribute to Phafin2's cell membrane binding. Phafin2 also contains a poly aspartic acid (polyD) motif in its C-terminal region that can specifically autoinhibit the PH domain binding to membrane phosphatidylinositol 3-phosphate (PtdIns3P). Firstly, the study investigated the domain-based evolutionary pattern of PH, FYVE, and polyD motif of Phafin2 among its orthologs and Phafin2- like proteins. Using different bioinformatics tools and resources, it was concluded that the polyD motif only evolved in Phafin2 and PH- or both PH-FYVE-containing proteins of animals, highlighting the association in cellular functions that might have evolved uniquely in animals. Moreover, PH domain-free FYVE-containing proteins lack polyD motifs. Secondly, intramolecular autoregulatory and membrane binding properties of Phafin2 were studied by employing liposome co-sedimentation assay, isothermal titration calorimetry, and nuclear magnetic resonance spectroscopy. The residues Gly38, Lys45, Leu45, Lys51, Ala52, and Arg53 of the PH domain form a positively charged binding pocket that can bind the negatively charged polyD motif. The mutated Phafin2 PH domain (K51A/R53C and R53C) was unable to bind to synthetic polyD peptides, establishing the significance of those residues for the interaction between the PH domain and polyD motif. Moreover, the study also concluded that Phafin2-mediated membrane binding is not curvature-dependent. / Master of Science / Phafin2 is a protein that plays a crucial role in several important cellular functions, including cell death, recycling of cellular components, and transporting materials within cells. The protein's ability to attach to cell membranes is mainly due to two of its specific regions, the PH and FYVE domains. Additionally, Phafin2 has a section called the polyD motif that can block the PH domain from binding to specific cell membrane molecules. This study explored how these regions of Phafin2 have evolved across different species, focusing on the PH, FYVE, and polyD motifs. The findings suggest that the polyD motif is unique to Phafin2 and similar animal proteins, potentially indicating a unique role in animal cell functions. Further experiments examined how Phafin2 regulates itself and binds to cell membranes. The study identified specific amino acids in the PH domain crucial for interacting with the polyD motif. When these amino acids were altered, Phafin2 could no longer bind to synthetic polyD peptides, highlighting their importance. Finally, the research determined that Phafin2's ability to bind to membranes does not depend on the shape or curvature of the membrane.

Phafin2 is a member of the Phafin protein family. Phafins are modular with an N-terminal PH (Pleckstrin Homology) domain followed by a central FYVE (Fab1, YOTB, Vac1, and EEA1) domain. Both the Phafin2 PH and FYVE domains bind phosphatidylinositol 3-phosphate [PtdIns(3)P], a phosphoinositide mainly found in endosomal and lysosomal membranes. Phafin2 acts as a PtdIns(3)P effector for endosomal cargo trafficking, macropinocytosis, apoptosis, and autophagy. The PtdIns(3)P binding activity is critical to the localization of Phafin2 on a specific membrane and, subsequently, helps the recruitment of other binding partners to the same membrane surface. However, there are no studies on the structural basis of PtdIns(3)P binding, the PtdIns(3)P-binding properties of each domain, and the apparent redundancy of two PtdIns(3)P binding domains in Phafin proteins. In the present dissertation, different biochemical and biophysical techniques were utilized to investigate the structural features of Phafin2 and its lipid interactions. This dissertation shows that Phafin2 is a moderately elongated monomer with a predicted α/β structure and ~40% random coil content. Phafin2 binds lipid bilayer-embedded PtdIns(3)P with high affinity; its PH and FYVE domains display distinct PtdIns(3)P-binding properties. Unlike the PH domain, the Phafin2 FYVE domain binds both membrane-embedded PtdIns(3)P and water-soluble dibutanoyl PtdIns(3)P with similar affinity. An intramolecular autoinhibition mechanism is found in Phafin2, in which a conserved C-terminal aspartic acid-rich (polyD) motif inhibits the binding of Phafin2 PH domain to PtdIns(3)P. The polyD motif specifically interacts with the Phafin2 PH domain. Using negative-stain Transmission Electron Microscopy, Phafin2 was found to cause membrane tubulation in a PtdIns(3)P-dependent manner. In conclusion, this study provides the structural and functional basis of Phafin2 lipid interactions and evidence of an intramolecular autoinhibition mechanism for PtdIns(3)P binding to the Phafin2 PH domain, which is mediated by the C-terminal polyD. The distinct PtdIns(3)P binding properties of the Phafin2 PH and FYVE domains may indicate that these two domains have different functions. Considering that the Phafin2 PH domain's PtdIns(3)P binding is intramolecularly regulated, cells may employ a unique mechanism to release the Phafin2 PH domain from the conserved C-terminal motif and control the functions of Phafin2 in PtdIns(3)P- and PH domain-dependent signaling pathways. / Doctor of Philosophy / Living cells need to absorb extracellular materials to sustain their growth and achieve cellular homeostasis. When cells require an uptake of liquids, they employ pinocytosis ("cell drinking"); when cells uptake solid particles, they use phagocytosis ("cell eating"); and when cells are in nutrient starvation status, they exploit an evolutionarily conserved process to survive known as autophagy ("self-eating"). Cells coordinate these activities through complex biochemical signaling systems. In each of these activities, a specific pathway is used to transfer the extracellular materials into the intracellular compartments and regulate the intracellular communications. Protein-lipid interactions are critical to these signaling pathways. This study focuses on the interactions between Phafin2 and phosphatidylinositol 3-phosphate [PtdIns(3)P]. Phafin2 is a cytoplasmic protein involved in autophagy, and PtdIns(3)P is a transient lipid signaling molecule localized to a specific organelle. After cells trigger autophagic events, Phafin2 protein molecules are associated with PtdIns(3)P. Subsequently, Phafin2 will recruit other protein binding partners. In this research project, biochemical and biophysical approaches were employed to study the structural features and PtdIns(3)P binding properties of Phafin2. Phafin2 was found to have two distinct PtdIns(3)P-binding domains; however, one of them is intramolecularly regulated. The results of this study help us to understand why Phafin2 displays two PtdIns(3)P-binding domains with different properties and how this is regulated, information that might be instrumental to understanding the roles of Phafin2 in physiological and disease scenarios.