Selective peptide inhibitors of bifunctional thymidylate synthase-dihydrofolate reductase from Toxoplasma gondii provide insights into domain–domain communication and allosteric regulation

Authors

  • Mark J. Landau,

    1. Department of Pharmacology, Yale University School of Medicine, New Haven, Connecticut
    2. Department of Molecular Biophysics and Biochemistry, Yale University School of Medicine, New Haven, Connecticut
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  • Hitesh Sharma,

    1. Department of Pharmacology, Yale University School of Medicine, New Haven, Connecticut
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  • Karen S. Anderson

    Corresponding author
    1. Department of Pharmacology, Yale University School of Medicine, New Haven, Connecticut
    2. Department of Molecular Biophysics and Biochemistry, Yale University School of Medicine, New Haven, Connecticut
    • Correspondence to: Karen S. Anderson, Yale University Medical School, Department of Pharmacology, 333 Cedar St., Rm SHM B350, New Haven, CT 06510. E-mail: karen.anderson@yale.edu

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  • The authors declare no conflict of interest.

Abstract

The bifunctional enzyme thymidylate synthase–dihydrofolate reductase (TS–DHFR) plays an essential role in DNA synthesis and is unique to several species of pathogenic protozoans, including the parasite Toxoplasma gondii. Infection by T. gondii causes the prevalent disease toxoplasmosis, for which TS–DHFR is a major therapeutic target. Here, we design peptides that target the dimer interface between the TS domains of bifunctional T. gondii TS–DHFR by mimicking β-strands at the interface, revealing a previously unknown allosteric target. The current study shows that these β-strand mimetic peptides bind to the apo-enzyme in a species-selective manner to inhibit both the TS and distal DHFR. Fluorescence spectroscopy was used to monitor conformational switching of the TS domain and demonstrate that these peptides induce a conformational change in the enzyme. Using structure-guided mutagenesis, nonconserved residues in the linker between TS and DHFR were identified that play a key role in domain–domain communication and in peptide inhibition of the DHFR domain. These studies validate allosteric inhibition of apo-TS, specifically at the TS–TS interface, as a potential target for novel, species-specific therapeutics for treating T. gondii parasitic infections and overcoming drug resistance.

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