Abstract
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 15N-labeled T2M4SOD1 was prepared from minimal media using 15NH4Cl as the 15N source. The T2M4SOD1 monomer (both 15N labeled and unlabeled) was correctly folded as confirmed by 1H-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 1H NMR and/or 19F-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, 1H-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 15N-NMR spectra of 15N-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.
Highlights
Amyotrophic Lateral Sclerosis (ALS) is a neurodegenerative disease that affects the motor neurons in the brain and spinal cord
There is evidence that the folding or misfolding of Cu/Zn superoxide dismutase 1 (SOD1) [3] and TDP-43 [4] may contribute to sALS, as aggregates of SOD1 have been found in post-mortem neural tissues of sALS individuals
The codons of the nucleotide sequence of T2M4SOD1 including the N-terminal 22 amino acids of the bacteriocuprein leader peptide (Fig 1B) were codon optimized for expression in E. coli and further optimized by removing any possible secondary RNA structures
Summary
Amyotrophic Lateral Sclerosis (ALS) is a neurodegenerative disease that affects the motor neurons in the brain and spinal cord. The loss of these motor neurons leads to an inability to move, walk, swallow, speak and, eventually, breathe. Proteins linked to fALS and sALS include the transactive response (TAR) DNA-binding protein 43 (TDP-43), an RNA binding protein called fused in sarcoma/translocated in liposarcoma and copper zinc superoxide dismutase (Cu/Zn SOD1). There is evidence that the folding or misfolding of Cu/Zn SOD1 [3] and TDP-43 [4] may contribute to sALS, as aggregates of SOD1 have been found in post-mortem neural tissues of sALS individuals. What is learned from studying fALS may aid in the understanding of sALS disease process
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