Abstract
Objectives
To determine the binding sites for l-phenylalanine in TyrR protein via a rational mutation analysis combining biosensors and computer-aided simulation.
Results
TyrR protein of Escherichia coli is the chief transcriptional regulator of several genes essential for the biosynthesis and transport of aromatic amino acids. The identification of ligand-binding sites is often the starting point for protein function annotation and structure-based protein design. Here we combined computer-aided prediction methods and biosensors to identify the ligand-binding sites for l-Phe in TyrR protein.
Conclusions
Residues at positions 160, 173 and 184 of TyrR protein are important for transcriptional activation of target genes tyrP induced by l-Phe, which indicates that they are the bona fide l-Phe binding sites of TyrR protein.
Similar content being viewed by others
References
Andrews AE, Dickson B, Lawley B, Cobbett C, Pittard AJ (1991) Importance of the position of TYR R boxes for repression and activation of the tyrP and aroF genes in Escherichia coli. J Bacteriol 173:5079–5085
Bordat A, Houvenaghel MC, German-Retana S (2015) Gibson assembly: an easy way to clone potyviral full-length infectious cDNA clones expressing an ectopic VPg. Virol J 12:89. https://doi.org/10.1186/s12985-015-0315-3
Deng Z et al (2015) TyrR, the regulator of aromatic amino acid metabolism, is required for mice infection of Yersinia pestis. Front Microbiol 6:110. https://doi.org/10.3389/fmicb.2015.00110
Glide (2018) Glide. Schrödinger, LLC, New York, p 2018
Koyanagi T, Katayama T, Suzuki H, Kumagai H (2008) Altered oligomerization properties of N316 mutants of Escherichia coli TyrR. J Bacteriol 190:8238–8243. https://doi.org/10.1128/jb.00889-08
Koyanagi T, Katayama T, Suzuki H, Onishi A, Yokozeki K, Kumagai H (2009) Hyperproduction of 3,4-dihydroxyphenyl-l-alanine (l-Dopa) using erwinia herbicola cells carrying a mutant transcriptional regulator TyrR. Biosci Biotech Bioch 73:1221–1223. https://doi.org/10.1271/bbb.90019
Liu-Smith F, Meyskens FL (2016) Molecular mechanisms of flavonoids in melanin synthesis and the potential for the prevention and treatment of melanoma. Mol Nutr Food Res 60:1264–1274. https://doi.org/10.1002/mnfr.201500822
Maestro (2018) Maestro. Schrödinger, LLC, New York, p 2018
Miller JH (1972) Experiments in molecular genetics. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY
SiteMap (2018) SiteMap. Schrödinger, LLC, New York, p 2018
Yang J, Camakaris H, Pittard AJ (1996) Further genetic analysis of the activation function of the TyrR regulatory protein of Escherichia coli. J Bacteriol 178:1120–1125. https://doi.org/10.1128/jb.178.4.1120-1125.1996
Yang J, Hwang JS, Camakaris H, Irawaty W, Ishihama A, Pittard J (2004) Mode of action of the TyrR protein: repression and activation of the tyrP promoter of Escherichia coli. Mol Microbiol 52:243–256. https://doi.org/10.1111/j.1365-2958.2003.03965.x
Yang J, Roy A, Zhang Y (2013) Protein-ligand binding site recognition using complementary binding-specific substructure comparison and sequence profile alignment. Bioinformatics 29:2588–2595. https://doi.org/10.1093/bioinformatics/btt447
Zhao D et al (2016) Development of a fast and easy method for Escherichia coli genome editing with CRISPR/Cas9. Microb Cell Fact 15:205. https://doi.org/10.1186/s12934-016-0605-5
Acknowledgements
This work was financially supported by National Key R&D Program of China (2018YFD0901001), Tianjin Science Fund for Distinguished Young Scholars (17JCJQJC45300), Nature Science Foundation of Tianjin City (CN) (16JCYBJC23500), Tianjin Science and Technology Project (15PTCYSY00020) and the Science and Technology Service Network (STS) Initiative of Chinese Academy of Sciences.
Supporting information
Supplementary Table 1—Strains and plasmids used in this study.
Supplementary Table 2—Primers used in this study.
Supplementary Figure 1—The effect of 184 site of TyrR on the binding mode of l-phe. The representative conformation of l-phe with 173 and 160 sites is verified by mutation S184L/V. The mutations S184L and S184V would clash with the benzene ring of l-Phe when binds 173 and 160 sites.
Supplementary Figure 2—Representative conformations of three clusters in MD simulations for 15 site of TyrR protein.
Author information
Authors and Affiliations
Corresponding authors
Ethics declarations
Conflict of interest
The authors declare that they have no conflict of interest.
Additional information
Publisher's Note
Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
Electronic supplementary material
Below is the link to the electronic supplementary material.
Rights and permissions
About this article
Cite this article
Bai, D., Ding, D., Li, J. et al. Pinpointing the l-phenylalanine binding sites of TyrR using biosensors and computer-aided simulation. Biotechnol Lett 41, 401–408 (2019). https://doi.org/10.1007/s10529-019-02645-x
Received:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1007/s10529-019-02645-x