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

Research output: Contribution to journal › Article › peer-review

1 Citation (Scopus)

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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
Volume	32
Issue number	7
Early online date	11 Mar 2025
DOIs	https://doi.org/10.1038/s41594-025-01516-6
Publication status	Published - Jul 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, 32(7), Article 2378. 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 - Loeff, Luuk

AU - Kroupova, Alena

AU - Asanović, Igor

AU - Boneberg, Franziska M.

AU - Pfleiderer, Moritz M.

AU - Riermeier, Luca

AU - Leitner, Alexander

AU - Ferdigg, Andrè

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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AN - SCOPUS:86000774168

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JO - Nature Structural and Molecular Biology

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

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