A discriminator code–based DTD surveillance ensures faithful glycine delivery for protein biosynthesis in bacteria

Abstract

D-aminoacyl-tRNA deacylase (DTD) acts on achiral glycine, in addition to D-amino acids, attached to tRNA. We have recently shown that this activity enables DTD to clear non-cognate Gly-tRNAAla with 1000-fold higher efficiency than its activity on Gly-tRNAGly, indicating tRNA-based modulation of DTD (Pawar et al., 2017). Here, we show that tRNA's discriminator base predominantly accounts for this activity difference and is the key to selection by DTD. Accordingly, the uracil discriminator base, serving as a negative determinant, prevents Gly-tRNAGly misediting by DTD and this protection is augmented by EF-Tu. Intriguingly, eukaryotic DTD has inverted discriminator base specificity and uses only G3•U70 for tRNAGly/Ala discrimination. Moreover, DTD prevents alanine-to-glycine misincorporation in proteins rather than only recycling mischarged tRNAAla. Overall, the study reveals the unique co-evolution of DTD and discriminator base, and suggests DTD's strong selection pressure on bacterial tRNAGlys to retain a pyrimidine discriminator code.

Data availability

Biochemical data is available as a source data file. All other data are included in the manuscript and supporting files.

Article and author information

Author details

  1. Santosh Kumar Kuncha

    CSIR-Centre for Cellular and Molecular Biology, Hyderabad, India
    Competing interests
    The authors declare that no competing interests exist.
  2. Katta Suma

    CSIR-Centre for Cellular and Molecular Biology, Hyderabad, India
    Competing interests
    The authors declare that no competing interests exist.
  3. Komal Ishwar Pawar

    CSIR-Centre for Cellular and Molecular Biology, Hyderabad, India
    Competing interests
    The authors declare that no competing interests exist.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0002-1968-9851
  4. Jotin Gogoi

    CSIR-Centre for Cellular and Molecular Biology, Hyderabad, India
    Competing interests
    The authors declare that no competing interests exist.
  5. Satya Brata Routh

    CSIR-Centre for Cellular and Molecular Biology, Hyderabad, India
    Competing interests
    The authors declare that no competing interests exist.
  6. Sambhavi Pottabathini

    CSIR-Centre for Cellular and Molecular Biology, Hyderabad, India
    Competing interests
    The authors declare that no competing interests exist.
  7. Shobha P Kruparani

    CSIR-Centre for Cellular and Molecular Biology, Hyderabad, India
    Competing interests
    The authors declare that no competing interests exist.
  8. Rajan Sankaranarayanan

    CSIR-Centre for Cellular and Molecular Biology, Hyderabad, India
    For correspondence
    sankar@ccmb.res.in
    Competing interests
    The authors declare that no competing interests exist.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0003-4524-9953

Funding

Department of Biotechnology , Ministry of Science and Technology (Centre of Excellence)

  • Rajan Sankaranarayanan

Science and Engineering Research Board (J. C. Bose Fellowship)

  • Rajan Sankaranarayanan

Department of Science and Technology, Ministry of Science and Technology (DST-INSPIRE)

  • Santosh Kumar Kuncha

The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.

Reviewing Editor

  1. Jonathan P Staley, University of Chicago, United States

Publication history

  1. Received: May 14, 2018
  2. Accepted: August 7, 2018
  3. Accepted Manuscript published: August 9, 2018 (version 1)
  4. Version of Record published: August 17, 2018 (version 2)

Copyright

© 2018, Kuncha et al.

This article is distributed under the terms of the Creative Commons Attribution License permitting unrestricted use and redistribution provided that the original author and source are credited.

Metrics

  • 1,497
    Page views
  • 159
    Downloads
  • 8
    Citations

Article citation count generated by polling the highest count across the following sources: Crossref, PubMed Central, Scopus.

Download links

A two-part list of links to download the article, or parts of the article, in various formats.

Downloads (link to download the article as PDF)

Open citations (links to open the citations from this article in various online reference manager services)

Cite this article (links to download the citations from this article in formats compatible with various reference manager tools)

  1. Santosh Kumar Kuncha
  2. Katta Suma
  3. Komal Ishwar Pawar
  4. Jotin Gogoi
  5. Satya Brata Routh
  6. Sambhavi Pottabathini
  7. Shobha P Kruparani
  8. Rajan Sankaranarayanan
(2018)
A discriminator code–based DTD surveillance ensures faithful glycine delivery for protein biosynthesis in bacteria
eLife 7:e38232.
https://doi.org/10.7554/eLife.38232

Further reading

    1. Biochemistry and Chemical Biology
    Mengyang Fan, Wenchao Lu ... Nathanael S Gray
    Research Article Updated

    The transcription factor TEAD, together with its coactivator YAP/TAZ, is a key transcriptional modulator of the Hippo pathway. Activation of TEAD transcription by YAP has been implicated in a number of malignancies, and this complex represents a promising target for drug discovery. However, both YAP and its extensive binding interfaces to TEAD have been difficult to address using small molecules, mainly due to a lack of druggable pockets. TEAD is post-translationally modified by palmitoylation that targets a conserved cysteine at a central pocket, which provides an opportunity to develop cysteine-directed covalent small molecules for TEAD inhibition. Here, we employed covalent fragment screening approach followed by structure-based design to develop an irreversible TEAD inhibitor MYF-03–69. Using a range of in vitro and cell-based assays we demonstrated that through a covalent binding with TEAD palmitate pocket, MYF-03–69 disrupts YAP-TEAD association, suppresses TEAD transcriptional activity and inhibits cell growth of Hippo signaling defective malignant pleural mesothelioma (MPM). Further, a cell viability screening with a panel of 903 cancer cell lines indicated a high correlation between TEAD-YAP dependency and the sensitivity to MYF-03–69. Transcription profiling identified the upregulation of proapoptotic BMF gene in cancer cells that are sensitive to TEAD inhibition. Further optimization of MYF-03–69 led to an in vivo compatible compound MYF-03–176, which shows strong antitumor efficacy in MPM mouse xenograft model via oral administration. Taken together, we disclosed a story of the development of covalent TEAD inhibitors and its high therapeutic potential for clinic treatment for the cancers that are driven by TEAD-YAP alteration.

    1. Biochemistry and Chemical Biology
    Lu Hu, Yang Sun ... Xu Wu
    Short Report Updated

    The TEA domain (TEAD) transcription factor forms a transcription co-activation complex with the key downstream effector of the Hippo pathway, YAP/TAZ. TEAD-YAP controls the expression of Hippo-responsive genes involved in cell proliferation, development, and tumorigenesis. Hyperactivation of TEAD-YAP activities is observed in many human cancers and is associated with cancer cell proliferation, survival, and immune evasion. Therefore, targeting the TEAD-YAP complex has emerged as an attractive therapeutic approach. We previously reported that the mammalian TEAD transcription factors (TEAD1–4) possess auto-palmitoylation activities and contain an evolutionarily conserved palmitate-binding pocket (PBP), which allows small-molecule modulation. Since then, several reversible and irreversible inhibitors have been reported by binding to PBP. Here, we report a new class of TEAD inhibitors with a novel binding mode. Representative analog TM2 shows potent inhibition of TEAD auto-palmitoylation both in vitro and in cells. Surprisingly, the co-crystal structure of the human TEAD2 YAP-binding domain (YBD) in complex with TM2 reveals that TM2 adopts an unexpected binding mode by occupying not only the hydrophobic PBP, but also a new side binding pocket formed by hydrophilic residues. RNA-seq analysis shows that TM2 potently and specifically suppresses TEAD-YAP transcriptional activities. Consistently, TM2 exhibits strong antiproliferation effects as a single agent or in combination with a MEK inhibitor in YAP-dependent cancer cells. These findings establish TM2 as a promising small-molecule inhibitor against TEAD-YAP activities and provide new insights for designing novel TEAD inhibitors with enhanced selectivity and potency.