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Cloning and high-level expression of monomeric human superoxide dismutase 1 (SOD1) and its interaction with pyrimidine analogs
Superoxide dismutase 1 (SOD1) is known to be involved in the pathogenesis of Amyotrophic Lateral Sclerosis (ALS) and is therefore considered to be an important ALS drug target. Identifying potential drug leads that bind to SOD1 and characterizing their interactions by nuclear magnetic resonance (NMR...
Autores principales: | , , , , |
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Formato: | Online Artículo Texto |
Lenguaje: | English |
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Public Library of Science
2021
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Materias: | |
Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7909654/ https://www.ncbi.nlm.nih.gov/pubmed/33635895 http://dx.doi.org/10.1371/journal.pone.0247684 |
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author | LeVatte, Marcia Lipfert, Matthias Roy, Dipankar Kovalenko, Andriy Wishart, David Scott |
author_facet | LeVatte, Marcia Lipfert, Matthias Roy, Dipankar Kovalenko, Andriy Wishart, David Scott |
author_sort | LeVatte, Marcia |
collection | PubMed |
description | Superoxide dismutase 1 (SOD1) is known to be involved in the pathogenesis of Amyotrophic Lateral Sclerosis (ALS) and is therefore considered to be an important ALS drug target. Identifying potential drug leads that bind to SOD1 and characterizing their interactions by nuclear magnetic resonance (NMR) spectroscopy is complicated by the fact that SOD1 is a homodimer. Creating a monomeric version of SOD1 could alleviate these issues. A specially designed monomeric form of human superoxide dismutase (T2M4SOD1) was cloned into E. coli and its expression significantly enhanced using a number of novel DNA sequence, leader peptide and growth condition optimizations. Uniformly (15)N-labeled T2M4SOD1 was prepared from minimal media using (15)NH(4)Cl as the (15)N source. The T2M4SOD1 monomer (both (15)N labeled and unlabeled) was correctly folded as confirmed by (1)H-NMR spectroscopy and active as confirmed by an in-gel enzymatic assay. To demonstrate the utility of this new SOD1 expression system for NMR-based drug screening, eight pyrimidine compounds were tested for binding to T2M4SOD1 by monitoring changes in their (1)H NMR and/or (19)F-NMR spectra. Weak binding to 5-fluorouridine (FUrd) was observed via line broadening, but very minimal spectral changes were seen with uridine, 5-bromouridine or trifluridine. On the other hand, (1)H-NMR spectra of T2M4SOD1 with uracil or three halogenated derivatives of uracil changed dramatically suggesting that the pyrimidine moiety is the crucial binding component of FUrd. Interestingly, no change in tryptophan 32 (Trp32), the putative receptor for FUrd, was detected in the (15)N-NMR spectra of (15)N-T2M4SOD1 when mixed with these uracil analogs. Molecular docking and molecular dynamic (MD) studies indicate that interaction with Trp32 of SOD1 is predicted to be weak and that there was hydrogen bonding with the nearby aspartate (Asp96), potentiating the Trp32-uracil interaction. These studies demonstrate that monomeric T2M4SOD1 can be readily used to explore small molecule interactions via NMR. |
format | Online Article Text |
id | pubmed-7909654 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2021 |
publisher | Public Library of Science |
record_format | MEDLINE/PubMed |
spelling | pubmed-79096542021-03-05 Cloning and high-level expression of monomeric human superoxide dismutase 1 (SOD1) and its interaction with pyrimidine analogs LeVatte, Marcia Lipfert, Matthias Roy, Dipankar Kovalenko, Andriy Wishart, David Scott PLoS One Research Article Superoxide dismutase 1 (SOD1) is known to be involved in the pathogenesis of Amyotrophic Lateral Sclerosis (ALS) and is therefore considered to be an important ALS drug target. Identifying potential drug leads that bind to SOD1 and characterizing their interactions by nuclear magnetic resonance (NMR) spectroscopy is complicated by the fact that SOD1 is a homodimer. Creating a monomeric version of SOD1 could alleviate these issues. A specially designed monomeric form of human superoxide dismutase (T2M4SOD1) was cloned into E. coli and its expression significantly enhanced using a number of novel DNA sequence, leader peptide and growth condition optimizations. Uniformly (15)N-labeled T2M4SOD1 was prepared from minimal media using (15)NH(4)Cl as the (15)N source. The T2M4SOD1 monomer (both (15)N labeled and unlabeled) was correctly folded as confirmed by (1)H-NMR spectroscopy and active as confirmed by an in-gel enzymatic assay. To demonstrate the utility of this new SOD1 expression system for NMR-based drug screening, eight pyrimidine compounds were tested for binding to T2M4SOD1 by monitoring changes in their (1)H NMR and/or (19)F-NMR spectra. Weak binding to 5-fluorouridine (FUrd) was observed via line broadening, but very minimal spectral changes were seen with uridine, 5-bromouridine or trifluridine. On the other hand, (1)H-NMR spectra of T2M4SOD1 with uracil or three halogenated derivatives of uracil changed dramatically suggesting that the pyrimidine moiety is the crucial binding component of FUrd. Interestingly, no change in tryptophan 32 (Trp32), the putative receptor for FUrd, was detected in the (15)N-NMR spectra of (15)N-T2M4SOD1 when mixed with these uracil analogs. Molecular docking and molecular dynamic (MD) studies indicate that interaction with Trp32 of SOD1 is predicted to be weak and that there was hydrogen bonding with the nearby aspartate (Asp96), potentiating the Trp32-uracil interaction. These studies demonstrate that monomeric T2M4SOD1 can be readily used to explore small molecule interactions via NMR. Public Library of Science 2021-02-26 /pmc/articles/PMC7909654/ /pubmed/33635895 http://dx.doi.org/10.1371/journal.pone.0247684 Text en © 2021 LeVatte et al http://creativecommons.org/licenses/by/4.0/ This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0/) , which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. |
spellingShingle | Research Article LeVatte, Marcia Lipfert, Matthias Roy, Dipankar Kovalenko, Andriy Wishart, David Scott Cloning and high-level expression of monomeric human superoxide dismutase 1 (SOD1) and its interaction with pyrimidine analogs |
title | Cloning and high-level expression of monomeric human superoxide dismutase 1 (SOD1) and its interaction with pyrimidine analogs |
title_full | Cloning and high-level expression of monomeric human superoxide dismutase 1 (SOD1) and its interaction with pyrimidine analogs |
title_fullStr | Cloning and high-level expression of monomeric human superoxide dismutase 1 (SOD1) and its interaction with pyrimidine analogs |
title_full_unstemmed | Cloning and high-level expression of monomeric human superoxide dismutase 1 (SOD1) and its interaction with pyrimidine analogs |
title_short | Cloning and high-level expression of monomeric human superoxide dismutase 1 (SOD1) and its interaction with pyrimidine analogs |
title_sort | cloning and high-level expression of monomeric human superoxide dismutase 1 (sod1) and its interaction with pyrimidine analogs |
topic | Research Article |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7909654/ https://www.ncbi.nlm.nih.gov/pubmed/33635895 http://dx.doi.org/10.1371/journal.pone.0247684 |
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