Crystallographic studies of ectonucleotide pyrophosphatase/phosphodiesterase 1
- The STING (STimulatory of INterferon Genes) pathway is an innate immune pathway that senses the presence of double-stranded DNA (dsDNA) in the cytosol. dsDNA binds to cyclic GMP/AMP synthase, which catalyzes the production of 2'3'-cyclic GMP/AMP (cGAMP), a small molecule. cGAMP then binds to the STING protein, leading to the production of Type 1 interferons and an immune response. Ectonucleotide pyrophosphatase/phosphodiesterase 1 (ENPP1) is an extracellular regulator of the STING pathway that hydrolyzes cGAMP to AMP and GMP, and acts as an innate immune checkpoint molecule. STF-1084 is an ENPP1 inhibitor we developed to further study ENPP1 activity in the cancer context. I generated a 3.2 Å crystal structure of ENPP1 in complex to STF-1084 to understand its mechanism of inhibition and guide subsequent inhibitor designs. Using this structural information, we were able to guide development of more potent ENPP1 inhibitors. To further understand how ENPP1 hydrolyzes cGAMP and explain why we can create substrate-specific ENPP1 mutants, I generated a 1.9 Å crystal structure of catalytically inactive bacterial Xac NPP in complex with linearized cGAMP. These data revealed no substantial interactions between the GMP of cGAMP and NPP side chains, contradicting proposals postulated by previous publications. We generated an NPP H214A mutant that degrades ATP but not cGAMP. Using these data in conjunction with structural data to understand cGAMP hydrolysis, we hypothesized that His214 coordinates a zinc ion that is necessary for leaving group stabilization in cGAMP hydrolysis but not ATP hydrolysis. To address this question, we solved a 2.0 Å structure of NPP H214A in complex with a non-hydrolyzable bisphosphorothioate-cGAMP analog. This structure revealed only one coordinated zinc ion in the active site, supporting the hypothesis that His214 is necessary for coordination of a leaving group-stabilizing zinc ion. To further study ENPP1 in various contexts, I attempted to generate macromolecular tools for a number of biochemical applications. Nanobodies are single chain macromolecules that recognize specific protein sequences and/or conformations. Nanobodies can be used as tools in a variety of applications, including X-ray crystallography, Western blotting, flow cytometry, and enzymatic studies. Using the Kruse yeast display nanobody library, I isolated 9 unique nanobody sequences that had some affinity for ENPP1 and characterized their ENPP1-binding affinity and their utility as macromolecular tools. This work enhances our understanding how ENPP1 functions as a hydrolase and provides new avenues for modulating ENPP1 activity.
|Type of resource
|electronic resource; remote; computer; online resource
|1 online resource.
|Brown, Jenifer Alexandra
|Kim, Peter, 1958-
|Kim, Peter, 1958-
|Degree committee member
|Stanford University, Biophysics Program
|Statement of responsibility
|Jenifer Alexandra Brown.
|Submitted to the Department of Biophysics Program.
|Thesis Ph.D. Stanford University 2021.
- © 2021 by Jenifer Alexandra Brown
- This work is licensed under a Creative Commons Attribution Non Commercial 3.0 Unported license (CC BY-NC).
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