Target of Myb 1 (TOM1) is an endosomal adaptor protein that plays a role in cargo membrane trafficking for degradation by serving as an alternative endosomal sorting complex required for transport component. TOM1 has also been shown to serve as a novel phosphatidylinositol 5-phosphate (PtdIns5P) effector at signaling endosomes through its VHS domain, delaying cargo degradation in a bacterial infection model. The aim of this thesis is to clarify the structural and functional basis of the autoregulation mechanism of TOM1 to switch from endosomal protein trafficking to the bacterial survival signaling pathway.
Our thermal denaturation and spectroscopic studies demonstrate that PtdIns5P reduced thermostability, interhelical contacts, and conformational compaction of TOM1 VHS. The thermodynamic studies indicate that TOM1 VHS endothermically binds to PtdIns5P through two potential noncooperative binding sites, with its acyl chains playing a relevant role in the interaction. These findings suggest that, under Shigella flexneri infection, TOM1 may interact with downstream effectors in a different VHS domain conformational state, thus involving the protein in bacterial survival signaling pathways.
In order to obtain molecular details for the interaction of the TOM1 VHS domain for PtdIns5P and Ubiquitin (Ub), the backbone assignment information was obtained by performing NMR experiments, which assigned backbone 1H, 13C, and 15N resonances of the TOM1 VHS domain. With this structural information, our heteronuclear single quantum coherence and molecular dynamics simulations data revealed that TOM1 VHS interacts with PtdIns5P following a fast-exchange regime, with the PtdIns5P binding site predicted to be at a region spanning α-helices 6 to 8. Further mutagenesis and lipid-protein overlay assay studies indicated that K147 plays a critical role in the binding of TOM1 VHS domain to PtdIns5P.
TOM1, unexpectedly, did not bind PtdIns5P. Using truncated forms of TOM1 protein, we discovered that neither TOM1 GAT domain nor the C-terminal domain modulated TOM1 VHS's PtdIns5P binding; however, surprisingly, a linker sequence between the TOM1 VHS and GAT domains exhibited an autoinhibition role for TOM1 binding to PtdIns5P. This linker region was observed to induce local conformational changes on the structure of TOM1 VHS domain, especially around α-helices 6 and 8, which are proposed to build up the binding pocket for PtdIns5P. In order to investigate whether the linker region between TOM1 VHS and GAT domain can also regulate the Ub association of TOM1 VHS domain, the binding properties of TOM1 and its domains to Ub were explored. Unexpectedly, the binding affinity of TOM1 VHS-linker for Ub was increased about 10-fold when compared with that for the TOM1 VHS domain, suggesting that the linker enhances the avidity of TOM1 for ubiquitinated cargo. Structural analysis indicated that the linker region may cap the conventional Ub-binding site of TOM1 VHS, thus forming a more compact structure.
In summary, this study uncovered a novel intramolecular modulatory mechanism in TOM1 that regulates ligand recognition by its VHS domain. By providing the molecular basis of the TOM1 interactions, we may provide cargo sorting mechanistic insights, create functionally specific mutations, and precisely manipulate TOM1 function under bacterial infection conditions, and other yet-to-be-discovered PtdIns5P-dependent signaling pathways. / Doctor of Philosophy / Membrane trafficking is a delivery network established in a cell to transport proteins (cargoes) from one intracellular place to another one to control their activity. TOM1 is a protein involved in this process, which plays a role in transporting cargoes for degradation. Defects in this trafficking pathway lead to human diseases, such as immunodeficiency and neurodegeneration diseases. TOM1 has also been shown to be beneficial for bacterial survival in human cells. However, how TOM1 switches its role form protein trafficking to bacterial pathogenesis is still unclear. In our study, we discovered an internal region of TOM1 may serve as a switch to shift the role of TOM1 in human cells. In an "on" status, TOM1 favors to transport cargoes, while in an "off" status, TOM1 is used for bacteria survival. This study provides insights in the function of TOM1 which is beneficial for the design of novel therapeutic strategies against TOM1, which will prevent the progress of bacterial infections.
| Identifer | oai:union.ndltd.org:VTETD/oai:vtechworks.lib.vt.edu:10919/112748 |
| Date | 08 June 2020 |
| Creators | Xiong, Wen |
| Contributors | Biological Sciences, Capelluto, Daniel G., Li, Jianyong, Jones, Caroline N., Yang, Zhaomin |
| Publisher | Virginia Tech |
| Source Sets | Virginia Tech Theses and Dissertation |
| Detected Language | English |
| Type | Dissertation |
| Format | ETD, application/pdf, application/pdf, application/pdf, application/pdf, application/pdf, application/pdf |
| Rights | This item is protected by copyright and/or related rights. Some uses of this item may be deemed fair and permitted by law even without permission from the rights holder(s), or the rights holder(s) may have licensed the work for use under certain conditions. For other uses you need to obtain permission from the rights holder(s). |
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