The T-cell lymphoma invasion and metastasis (Tiam) family of proteins are guanine nucleotide exchange factors (GEFs). The Tiam family has two homologs, Tiam1 and Tiam2, which activate the Rho-family GTPase Rac1 and are crucial for cell-cell adhesion. Tiam1/2 contain several protein-protein interaction domains, in particular a PDZ domain. PDZ domains are distributed from C. elegans to H. sapiens proteomes. The prototypical ligands of PDZ domains are specific sequences located at the C-termini of their binding partners. In this thesis I examined the specificity of wild-type (WT) and two mutant forms of the Tiam1/2 PDZ domains, which bind to three ligands derived from adhesion receptors - Syndecan1 (SDC1), Caspr4 and Neurexin1 (NRXN1).
The specificity of the Tiam1 PDZ WT domain for individual SDC family members was identified and found to be determined by distinct electrostatic interactions. Based on X-ray crystallographic studies, two ligand binding pockets were crucial for SDC1 binding while an additional pocket was important for accommodating the phosphate group in phosphorylated SDC1 (pSDC1). Methyl relaxation experiments of PDZ/SDC1 and PDZ/pSDC1 complexes revealed that PDZ-phosphoryl interactions dampened dynamic motions in a distal region of the PDZ domain by decoupling them from the ligand-binding site. Our data suggest a selection model by which specificity and phosphorylation regulate PDZ/SDC interactions and signaling events.
Previous studies showed that the Tiam1 and Tiam2 WT PDZ domains have distinct ligand specificities. Moreover, a mutant of the Tiam1 PDZ domain that has four non-conserved residues substituted to the analogous residues in the Tiam2 PDZ domain (designated QM) showed switched specificity. Here, I determined the crystal structures of the free and ligand-bound Tiam1 PDZ QM. These structures revealed that two enlarged ligand binding pockets and a favorable electrostatic interaction are critical for the changed specificity. Complementary NMR studies showed enhanced dynamic motions in the Tiam1 PDZ QM domain. These data support a conformational selection model where mutations in the PDZ domain lead to the specificity switch through structural changes in binding pockets and enhanced dynamic motions.
The Tiam2 PDZ QM domain was produced by substituting the four specificity residues to those in the Tiam1 PDZ WT domain. The Tiam2 PDZ QM had a switched specificity, similar to that found in the Tiam1 PDZ WT. NMR-based HSQC spectra and quantitative relaxation studies revealed that the Tiam2 PDZ WT domain had dynamic motions in several loop regions, while motions in the QM PDZ domain were extended to additional regions. Thermodynamic analyses of Tiam2 PDZ mutated domains uncovered energetic cooperativity between two binding pockets, with respect to both ligand binding and protein folding. I propose that the altered dynamic and thermodynamic properties support the ligand binding specificity switch in the Tiam2 PDZ QM.
The literature indicates that aberrant SDC1 and Tiam1-Rac1 signaling pathways contribute to tumor progression. These two pathways are coupled by the PDZ-mediated Tiam1/SDC1 interaction. Here, I integrated structure-based in silico docking and an unbiased high throughput screen to discover inhibitors targeting this PDZ-mediated interaction and in order to inhibit downstream Tiam1-Rac1 signaling and tumor progression. Several compounds were identified and validated to block this interaction and downstream Rac1 signaling. Further modification of these molecules could lead to inhibitors with more potency and thus therapeutic values.
In summary, detailed biochemical, structural and dynamics studies were performed to understand the mechanistic origins of ligand specificity in the Tiam1 and Tiam2 PDZ domains. These results highlight the significant structure-dynamics-function relationship in the PDZ domain and imply that rewiring these interactions is possible. Moreover, a high throughput screen identified small molecule inhibitors targeting these PDZ domains. These inhibitors could be used as molecular probes in cells to dissect the physiological roles of signaling cascades controlled by Tiam GEFs.
| Identifer | oai:union.ndltd.org:uiowa.edu/oai:ir.uiowa.edu:etd-8104 |
| Date | 01 December 2014 |
| Creators | Liu, Xu |
| Contributors | Fuentes, Ernesto Jorge, 1966- |
| Publisher | University of Iowa |
| Source Sets | University of Iowa |
| Language | English |
| Detected Language | English |
| Type | dissertation |
| Format | application/pdf |
| Source | Theses and Dissertations |
| Rights | Copyright © 2014 Xu Liu |
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