Structural Control of Nonnative Ligand Binding in Engineered Mutants of Phosphoenolpyruvate Carboxykinase

Tang, Henry Y.  H.; Shin, David S.; Hura, Greg L.; Yang, Yue; Hu, Xiaoyu; Lightstone, Felice C.; McGee, Matthew D.; Padgett, Hal S.; Yannone, Steven M.; Tainer, John A.

doi:10.1021/acs.biochem.8b00963

Title: Structural Control of Nonnative Ligand Binding in Engineered Mutants of Phosphoenolpyruvate Carboxykinase

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Abstract

Protein engineering to alter recognition underlying ligand binding and activity has enormous potential. Here, ligand binding for Escherichia coli phosphoenolpyruvate carboxykinase (PEPCK), which converts oxaloacetate into CO₂ and phosphoenolpyruvate as the first committed step in gluconeogenesis, was engineered to accommodate alternative ligands as an exemplary system with structural information. From our identification of bicarbonate binding in the PEPCK active site at the supposed CO₂ binding site, we probed binding of nonnative ligands with three oxygen atoms arranged to resemble the bicarbonate geometry. Crystal structures of PEPCK and point mutants with bound nonnative ligands thiosulfate and methanesulfonate along with strained ATP and reoriented oxaloacetate intermediates and unexpected bicarbonate were determined and analyzed. The mutations successfully altered the bound ligand position and orientation and its specificity: mutated PEPCKs bound either thiosulfate or methanesulfonate but never both. Computational calculations predicted a methanesulfonate binding mutant and revealed that release of the active site ordered solvent exerts a strong influence on ligand binding. Besides nonnative ligand binding, one mutant altered the Mn²⁺ coordination sphere: instead of the canonical octahedral ligand arrangement, the mutant in question had an only five-coordinate arrangement. From this work, critical features of ligand binding, position, and metal ion cofactor geometry requiredmore »« less

Authors:

Tang, Henry Y. H. ^[1]; Shin, David S. ^[2]; Hura, Greg L. ^[3]; Yang, Yue ^[4]; Hu, Xiaoyu ^[5]; Lightstone, Felice C. ^[4]; McGee, Matthew D. ^[6]; Padgett, Hal S. ^[6]; Yannone, Steven M. ^[2];

^[7]

Lawrence Berkeley National Lab. (LBNL), Berkeley, CA (United States). Molecular Biophysics and Integrated Bioimaging Division; Univ. of California, Berkeley, CA (United States). Dept. of Chemistry
Lawrence Berkeley National Lab. (LBNL), Berkeley, CA (United States). Molecular Biophysics and Integrated Bioimaging Division
Lawrence Berkeley National Lab. (LBNL), Berkeley, CA (United States). Molecular Biophysics and Integrated Bioimaging Division; Univ. of California, Santa Cruz, CA (United States). Dept. of Biochemistry and Chemistry
Lawrence Livermore National Lab. (LLNL), Livermore, CA (United States)
Tsinghua Univ., Beijing (China). Dept. of Chemical Engineering
Novici Biotech LLC, Vacaville, CA (United States)
Lawrence Berkeley National Lab. (LBNL), Berkeley, CA (United States). Molecular Biophysics and Integrated Bioimaging Division; Univ. of Texas, Houston, TX (United States). M. D. Anderson Cancer Center, Dept. of Molecular and Cellular Oncology

Publication Date:: Tue Oct 30 00:00:00 EDT 2018

Research Org.:: Lawrence Livermore National Lab. (LLNL), Livermore, CA (United States)

Sponsoring Org.:: USDOE Office of Science (SC), Biological and Environmental Research (BER); USDOE Advanced Research Projects Agency - Energy (ARPA-E)

OSTI Identifier:: 1513118

Report Number(s):: LLNL-JRNL-735122
Journal ID: ISSN 0006-2960; 885823

Grant/Contract Number:: AC52-07NA27344

Resource Type:: Accepted Manuscript

Journal Name:: Biochemistry

Additional Journal Information:: Journal Volume: 57; Journal Issue: 48; Journal ID: ISSN 0006-2960

Publisher:: American Chemical Society (ACS)

Country of Publication:: United States

Language:: English

Subject:: 59 BASIC BIOLOGICAL SCIENCES

Citation Formats


                    Tang, Henry Y.  H., Shin, David S., Hura, Greg L., Yang, Yue, Hu, Xiaoyu, Lightstone, Felice C., McGee, Matthew D., Padgett, Hal S., Yannone, Steven M., and Tainer, John A. Structural Control of Nonnative Ligand Binding in Engineered Mutants of Phosphoenolpyruvate Carboxykinase.  United States: N. p., 2018. 
Web.  doi:10.1021/acs.biochem.8b00963.

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                    Tang, Henry Y.  H., Shin, David S., Hura, Greg L., Yang, Yue, Hu, Xiaoyu, Lightstone, Felice C., McGee, Matthew D., Padgett, Hal S., Yannone, Steven M., & Tainer, John A. Structural Control of Nonnative Ligand Binding in Engineered Mutants of Phosphoenolpyruvate Carboxykinase.  United States.  https://doi.org/10.1021/acs.biochem.8b00963

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                    Tang, Henry Y.  H., Shin, David S., Hura, Greg L., Yang, Yue, Hu, Xiaoyu, Lightstone, Felice C., McGee, Matthew D., Padgett, Hal S., Yannone, Steven M., and Tainer, John A. Tue .  
"Structural Control of Nonnative Ligand Binding in Engineered Mutants of Phosphoenolpyruvate Carboxykinase".  United States.  https://doi.org/10.1021/acs.biochem.8b00963.  https://www.osti.gov/servlets/purl/1513118.

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@article{osti_1513118,

  title        = {Structural Control of Nonnative Ligand Binding in Engineered Mutants of Phosphoenolpyruvate Carboxykinase},

  author       = {Tang, Henry Y.  H. and Shin, David S. and Hura, Greg L. and Yang, Yue and Hu, Xiaoyu and Lightstone, Felice C. and McGee, Matthew D. and Padgett, Hal S. and Yannone, Steven M. and Tainer, John A.},

  abstractNote = {Protein engineering to alter recognition underlying ligand binding and activity has enormous potential. Here, ligand binding for Escherichia coli phosphoenolpyruvate carboxykinase (PEPCK), which converts oxaloacetate into CO2 and phosphoenolpyruvate as the first committed step in gluconeogenesis, was engineered to accommodate alternative ligands as an exemplary system with structural information. From our identification of bicarbonate binding in the PEPCK active site at the supposed CO2 binding site, we probed binding of nonnative ligands with three oxygen atoms arranged to resemble the bicarbonate geometry. Crystal structures of PEPCK and point mutants with bound nonnative ligands thiosulfate and methanesulfonate along with strained ATP and reoriented oxaloacetate intermediates and unexpected bicarbonate were determined and analyzed. The mutations successfully altered the bound ligand position and orientation and its specificity: mutated PEPCKs bound either thiosulfate or methanesulfonate but never both. Computational calculations predicted a methanesulfonate binding mutant and revealed that release of the active site ordered solvent exerts a strong influence on ligand binding. Besides nonnative ligand binding, one mutant altered the Mn2+ coordination sphere: instead of the canonical octahedral ligand arrangement, the mutant in question had an only five-coordinate arrangement. From this work, critical features of ligand binding, position, and metal ion cofactor geometry required for all downstream events can be engineered with small numbers of mutations to provide insights into fundamental underpinnings of protein–ligand recognition. Finally, through structural and computational knowledge, the combination of designed and random mutations aids in the robust design of predetermined changes to ligand binding and activity to engineer protein function.},

  doi          = {10.1021/acs.biochem.8b00963},

  journal      = {Biochemistry},

  number       = 48,

  volume       = 57,

  place        = {United States},

  year         = {Tue Oct 30 00:00:00 EDT 2018},

  month        = {Tue Oct 30 00:00:00 EDT 2018}

}

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