(681e) Computational Protein Design Targeting Oxidized RNA Modifications | AIChE

(681e) Computational Protein Design Targeting Oxidized RNA Modifications


Orr, A. A. - Presenter, Texas A&M University
Engels, S. M., University of Texas at Austin
Jakubowski, J. M., Texas A&M University
Woodcock, B. C., Texas A&M University
Contreras, L., The University of Texas at Austin
Tamamis, P., Texas A&M University
Modified RNAs are emerging key components in the field of post-transcriptional regulation of gene expression [1]. One important aspect on modified RNAs is their ability to bind to proteins, modulate RNA-protein interactions, thereby affecting cell function and regulation [2,3]. We recently demonstrated how computational methods can be used to study and predict the RNA modifications that can be recognized by a protein, using RNA binding to E. coli Polynucleotide Phosphorylase (PNPase) as a test case [4]. Here, we aimed to solve the inverse problem, i.e., to redesign E. coli PNPase to bind a key oxidized RNA modification, 8-hydroxygruanine (8-oxoG) with stronger affinity than the native protein.

Oxidized RNAs can lead to loss of normal protein function and have been implicated in several neurological disorders and cancers [5]. A few RNA binding proteins have been shown to reduce oxidized RNA and protect cells against oxidative stress. PNPase is one such RNA binding protein, and has been shown to bind 8-oxoG with high affinity and be key in preventing oxidative damage [6].

To address our aim, we used the modeled structure of E. coli PNPase binding to an 8-oxoG containing RNA strand derived from our previous study [4] and followed a two-stage procedure. In the first stage, we performed short simulations in CHARMM, introducing variable combinations of amino acids at the protein, and screened our energetically unfavorable amino acid combinations; amino acids introduction was guided by bioinformatics and the use biological constrained mutations. In the second stage, we used multiple multi-ns simulations in CHARMM and physics-based free energy calculations to identify the most promising PNPase designed variants with improved relative affinity to the native protein. Our results provide detailed energetic and structural insights into the role of the designed PNPase amino acids and their interactions with 8-oxoG, and their role in binding, and will be presented in tandem with experimental results. Our studies offer key biological insights into species which potentially possess high affinity to recognize oxidized (8-oxoG) RNA modifications. In addition, such studies can be used as a prototype for the redesign of proteins to recognize specific RNA modifications with high affinity, as novel potential agents for the prevention, detection and treatment of diseases.

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[2] Baldridge KC, Contreras LM. Functional implications of ribosomal RNA methylation in response to environmental stress. Crit Rev Biochem Mol Biol. 2014;49(1):69-89.

[3] Mihailovic MK, Chen A, Gonzalez-Rivera JC, Contreras LM. Defective Ribonucleoproteins, Mistakes in RNA Processing, and Diseases. Biochemistry. 2017;56(10):1367-1382.

[4] Orr AA, Gonzalez-Rivera JC, Wilson M, Bhikha PR, Wang D, Contreras LM, Tamamis P. A high-throughput and rapid computational method for screening of RNA post-transcriptional modifications that can be recognized by target proteins. Methods. 2018;143:34-47.

[5] Nunomura A, Honda K, Takeda A, Hirai K, Zhu X, Smith MA, Perry G. Oxidative Damage to RNA in Neurodegenerative Diseases. Journal of Biomedicine and Biotechnology. 2006;1-6.

[6] Wu J, Jiang Z, Liu M, et al. Polynucleotide phosphorylase protects Escherichia coli against oxidative stress. Biochemistry. 2009;48(9):2012-2020.