Mechanistic basis for PYROXD1-mediated protection of the human tRNA ligase complex against oxidative inactivation

Luuk Loeff; Alena Kroupova; Igor Asanović; Franziska M. Boneberg; Moritz M. Pfleiderer; Luca Riermeier; Alexander Leitner; Andrè Ferdigg; Fabian Ackle; Javier Martinez; Martin Jinek

doi:10.1038/s41594-025-01516-6

Mechanistic basis for PYROXD1-mediated protection of the human tRNA ligase complex against oxidative inactivation

Luuk Loeff, Alena Kroupova, Igor Asanović, Franziska M. Boneberg, Moritz M. Pfleiderer, Luca Riermeier, Alexander Leitner, Andrè Ferdigg, Fabian Ackle, Javier Martinez, Martin Jinek (Lead / Corresponding author)

Centre for Targeted Protein Degradation

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Abstract

The metazoan tRNA ligase complex (tRNA-LC) has essential roles in tRNA biogenesis and unfolded protein response. Its catalytic subunit RTCB contains a conserved active-site cysteine that is susceptible to metal ion-induced oxidative inactivation. The flavin-containing oxidoreductase PYROXD1 preserves the activity of human tRNA-LC in a NAD(P)H-dependent manner, but its protective mechanism remains elusive. Here, we report a cryogenic electron microscopic structure of the human RTCB–PYROXD1 complex, revealing that PYROXD1 directly interacts with the catalytic center of RTCB through its carboxy-terminal tail. NAD(P)H binding and FAD reduction allosterically control PYROXD1 activity and RTCB recruitment, while reoxidation of PYROXD1 enables timed release of RTCB. PYROXD1 interaction is mutually exclusive with Archease-mediated RTCB guanylylation, and guanylylated RTCB is intrinsically protected from oxidative inactivation. Together, these findings provide a mechanistic framework for the protective function of PYROXD1 that maintains the activity of the tRNA-LC under aerobic conditions.

Original language	English
Article number	2378
Number of pages	28
Journal	Nature Structural and Molecular Biology
Early online date	11 Mar 2025
DOIs	https://doi.org/10.1038/s41594-025-01516-6
Publication status	E-pub ahead of print - 11 Mar 2025

ASJC Scopus subject areas

Structural Biology
Molecular Biology

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Loeff, L., Kroupova, A., Asanović, I., Boneberg, F. M., Pfleiderer, M. M., Riermeier, L., Leitner, A., Ferdigg, A., Ackle, F., Martinez, J., & Jinek, M. (2025). Mechanistic basis for PYROXD1-mediated protection of the human tRNA ligase complex against oxidative inactivation. Nature Structural and Molecular Biology, Article 2378. Advance online publication. https://doi.org/10.1038/s41594-025-01516-6

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title = "Mechanistic basis for PYROXD1-mediated protection of the human tRNA ligase complex against oxidative inactivation",

abstract = "The metazoan tRNA ligase complex (tRNA-LC) has essential roles in tRNA biogenesis and unfolded protein response. Its catalytic subunit RTCB contains a conserved active-site cysteine that is susceptible to metal ion-induced oxidative inactivation. The flavin-containing oxidoreductase PYROXD1 preserves the activity of human tRNA-LC in a NAD(P)H-dependent manner, but its protective mechanism remains elusive. Here, we report a cryogenic electron microscopic structure of the human RTCB–PYROXD1 complex, revealing that PYROXD1 directly interacts with the catalytic center of RTCB through its carboxy-terminal tail. NAD(P)H binding and FAD reduction allosterically control PYROXD1 activity and RTCB recruitment, while reoxidation of PYROXD1 enables timed release of RTCB. PYROXD1 interaction is mutually exclusive with Archease-mediated RTCB guanylylation, and guanylylated RTCB is intrinsically protected from oxidative inactivation. Together, these findings provide a mechanistic framework for the protective function of PYROXD1 that maintains the activity of the tRNA-LC under aerobic conditions.",

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AU - Pfleiderer, Moritz M.

AU - Riermeier, Luca

AU - Leitner, Alexander

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AU - Ackle, Fabian

AU - Martinez, Javier

AU - Jinek, Martin

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N2 - The metazoan tRNA ligase complex (tRNA-LC) has essential roles in tRNA biogenesis and unfolded protein response. Its catalytic subunit RTCB contains a conserved active-site cysteine that is susceptible to metal ion-induced oxidative inactivation. The flavin-containing oxidoreductase PYROXD1 preserves the activity of human tRNA-LC in a NAD(P)H-dependent manner, but its protective mechanism remains elusive. Here, we report a cryogenic electron microscopic structure of the human RTCB–PYROXD1 complex, revealing that PYROXD1 directly interacts with the catalytic center of RTCB through its carboxy-terminal tail. NAD(P)H binding and FAD reduction allosterically control PYROXD1 activity and RTCB recruitment, while reoxidation of PYROXD1 enables timed release of RTCB. PYROXD1 interaction is mutually exclusive with Archease-mediated RTCB guanylylation, and guanylylated RTCB is intrinsically protected from oxidative inactivation. Together, these findings provide a mechanistic framework for the protective function of PYROXD1 that maintains the activity of the tRNA-LC under aerobic conditions.

AB - The metazoan tRNA ligase complex (tRNA-LC) has essential roles in tRNA biogenesis and unfolded protein response. Its catalytic subunit RTCB contains a conserved active-site cysteine that is susceptible to metal ion-induced oxidative inactivation. The flavin-containing oxidoreductase PYROXD1 preserves the activity of human tRNA-LC in a NAD(P)H-dependent manner, but its protective mechanism remains elusive. Here, we report a cryogenic electron microscopic structure of the human RTCB–PYROXD1 complex, revealing that PYROXD1 directly interacts with the catalytic center of RTCB through its carboxy-terminal tail. NAD(P)H binding and FAD reduction allosterically control PYROXD1 activity and RTCB recruitment, while reoxidation of PYROXD1 enables timed release of RTCB. PYROXD1 interaction is mutually exclusive with Archease-mediated RTCB guanylylation, and guanylylated RTCB is intrinsically protected from oxidative inactivation. Together, these findings provide a mechanistic framework for the protective function of PYROXD1 that maintains the activity of the tRNA-LC under aerobic conditions.

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Mechanistic basis for PYROXD1-mediated protection of the human tRNA ligase complex against oxidative inactivation

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