The genus Phytophthora contains a large number of species that are known plant pathogens of a variety of important crops. Phytophthora sojae, a hemibiotroph, causes approximately 1-2 billion dollars (US) of lost soybean world-wide each year. P. infestans, the causative agent of the Irish potato famine, is responsible for over 5 billion dollars (US) worth of lost potato each year. These destructive plant pathogens facilitate pathogenesis through the use of small secreted proteins known as effector proteins. A large subset of effector proteins is able to translocate into host cells and target plant defense pathways. P. sojae Avr1b is able to suppress cell death triggered by BAX and hydrogen peroxide. The W-domain of Avr1b is responsible for this functionality, and is recognized by the Rps1b gene product to induce effector triggered immunity. These oomycete effector proteins translocate into host cells via a highly conserved N-terminal motif known as RXLR-dEER without the use of any pathogen encoded machinery. In fungi an RXLR-like motif exists, [R,K,H] X [L,F,Y,M,~I] X, that is able to facilitate translocation without pathogen encoded machinery. Both functional RXLR and RXLR-like motifs are able to bind phosphatidylinositol-3-phosphate (PtdIns- 3-P) to mediate entry into host cells. The use of novel inhibitory mechanisms has shown effector entry can be blocked either by sequestering PtdIns-3-P on the outer leaflet of plant and animal cells or by competitive inhibition of the binding pocket of the RXLR or RXLR-like motifs. / Ph. D.

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.

Oomycetes, such as Phytophthora sojae, are plant pathogens that employ protein effectors that enter host cells to facilitate infection. Plants may overcome infection by recognizing pathogen effectors via intracellular receptors (R proteins) that form part of their defense system. Entry of some effector proteins into plant cells is mediated by conserved RxLR motifs in the effectors and phosphoinositides (PIPs) resident in the host plasma membrane such as phosphatidylinositol 3-phosphate (PtdIns(3)P). Recent reports differ regarding the regions on RxLR effector proteins involved in PIP recognition. To clarify these differences, I have structurally and functionally characterized the P. sojae effector, avirulence homolog-5 (Avh5). Using NMR spectroscopy, I demonstrate that Avh5 is helical in nature with a long N-terminal disordered region. Heteronuclear single quantum coherence titrations of Avh5 with the PtdIns(3)P head group, inositol 1,3-bisphosphate (Ins(1,3)P2), allowed us to identify a C-terminal lysine-rich helical region (helix 2) as the principal lipid-binding site in the protein, with the N-terminal RxLR (RFLR) motif playing a more minor role. Furthermore, mutations in the RFLR motif slightly affected PtdIns(3)P binding, while mutations in the basic helix almost abolished it. Avh5 exhibited moderate affinity for PtdIns(3)P, which increased the thermal stability of the protein. Mutations in the RFLR motif or in the basic region of Avh5 both significantly reduced protein entry into plant and human cells. Both regions independently mediated cell entry via a PtdIns(3)P-dependent mechanism. My findings support a model in which Avh5 transiently interacts with PtdIns(3)P by electrostatic interactions mainly through its positively charged helix 2 region, providing stability to the protein during RFLR-mediated host entry. / Ph. D.

Adaptor proteins, like Tollip and Tom1, facilitate cellular cargo sorting through their ubiquitin-binding domains. Tollip and Tom1 bind to each other through their TBD and GAT domains, respectively, whereas Tollip interacts with phosphatidylinositol-3-phosphate (PtdIns(3)P)-containing endosomal membranes. Tom1 and Tollip interaction and association with endosomes is proposed to be involved in the lysosomal degradation of polyubiquitinated cargo. Through cellular, biochemical, and biophysical techniques, we have further characterized the association of Tom1 with Tollip. Mutations in the binding interface of the Tom1 GAT and Tollip TBD complex leads to a subcellular mis-localization of both proteins, indicating that Tom1 may serve to direct Tollip to specific cellular pathways. It was determined that Tom1 inhibits the binding of Tollip to PtdIns(3)P and inhibition was reversed when mutations in the binding interface of the Tom1 GAT and Tollip TBD were present. Furthermore, it was established that, upon the binding of Tollip TBD to Tom1 GAT, ubiquitin is inhibited from binding to Tom1 GAT. It was also demonstrated that Tom1 GAT, but not Tollip TBD, can weakly bind to PtdIns(3)P. Consequently, we propose that association of Tom1 may serve to direct Tollip for involvement in specific cell signaling pathways. Gaining insight into the function of Tom1 and Tollip may lead to their use as therapeutic targets for increasing the efficiency of cargo trafficking and also for patients recovering from various cardiac injuries. / Master of Science