Integrative modeling reveals the molecular architecture of the intraflagellar transport A (IFT-A) complex

  1. Caitlyn L McCafferty  Is a corresponding author
  2. Ophelia Papoulas
  3. Mareike A Jordan
  4. Gabriel Hoogerbrugge
  5. Candice Nichols
  6. Gaia Pigino
  7. David W Taylor
  8. John B Wallingford
  9. Edward M Marcotte  Is a corresponding author
  1. The University of Texas at Austin, United States
  2. Max Planck Institute of Molecular Cell Biology and Genetics, Germany
  3. Human Technopole, Italy

Abstract

Intraflagellar transport (IFT) is a conserved process of cargo transport in cilia that is essential for development and homeostasis in organisms ranging from algae to vertebrates. In humans, variants in genes encoding subunits of the cargo-adapting IFT-A and IFT-B protein complexes are a common cause of genetic diseases known as ciliopathies. While recent progress has been made in determining the atomic structure of IFT-B, little is known of the structural biology of IFT-A. Here, we combined chemical cross-linking mass spectrometry and cryo-electron tomography with AlphaFold2-based prediction of both protein structures and interaction interfaces to model the overall architecture of the monomeric six-subunit IFT-A complex, as well as its polymeric assembly within cilia. We define monomer-monomer contacts and membrane-associated regions available for association with transported cargo, and we also use this model to provide insights into the pleiotropic nature of human ciliopathy-associated genetic variants in genes encoding IFT-A subunits. Our work demonstrates the power of integration of experimental and computational strategies both for multi-protein structure determination and for understanding the etiology of human genetic disease.

Data availability

Mass spectrometry proteomics data was deposited in the MassIVE/ProteomeXchange database (113) under accession number PXD032818. Cryo-tomography data was deposited in the Electron Microscopy Data Bank (114) under accession number EMD-26791. IFT-A models were deposited in the PDB-Dev database (115) as well as on Zenodo at doi: 10.5281/zenodo.7222413, along with additional supporting materials, including integrative modeling data and code.

Article and author information

Author details

  1. Caitlyn L McCafferty

    Department of Molecular Biosciences, The University of Texas at Austin, Austin, United States
    For correspondence
    clmccafferty@utexas.edu
    Competing interests
    The authors declare that no competing interests exist.
  2. Ophelia Papoulas

    Department of Molecular Biosciences, The University of Texas at Austin, Austin, United States
    Competing interests
    The authors declare that no competing interests exist.
  3. Mareike A Jordan

    Max Planck Institute of Molecular Cell Biology and Genetics, Dresden, Germany
    Competing interests
    The authors declare that no competing interests exist.
  4. Gabriel Hoogerbrugge

    Department of Molecular Biosciences, The University of Texas at Austin, Austin, United States
    Competing interests
    The authors declare that no competing interests exist.
  5. Candice Nichols

    Department of Molecular Biosciences, The University of Texas at Austin, Austin, United States
    Competing interests
    The authors declare that no competing interests exist.
  6. Gaia Pigino

    Human Technopole, Milan, Italy
    Competing interests
    The authors declare that no competing interests exist.
  7. David W Taylor

    Department of Molecular Biosciences, The University of Texas at Austin, Austin, United States
    Competing interests
    The authors declare that no competing interests exist.
  8. John B Wallingford

    Department of Molecular Biosciences, The University of Texas at Austin, Austin, United States
    Competing interests
    The authors declare that no competing interests exist.
  9. Edward M Marcotte

    Department of Molecular Biosciences, The University of Texas at Austin, Austin, United States
    For correspondence
    marcotte@utexas.edu
    Competing interests
    The authors declare that no competing interests exist.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0001-8808-180X

Funding

National Science Foundation (2019238253)

  • Caitlyn L McCafferty

National Institute of General Medical Sciences (R35GM122480)

  • Edward M Marcotte

National Institute of General Medical Sciences (R35GM138348)

  • David W Taylor

National Institute of Child Health and Human Development (HD085901)

  • John B Wallingford
  • Edward M Marcotte

Army Research Office (W911NF-12-1-0390)

  • Edward M Marcotte

Welch Foundation (F-1515)

  • Edward M Marcotte

Welch Foundation (F-1938)

  • David W Taylor

Max Planck Society

  • Mareike A Jordan
  • Gaia Pigino

Cancer Prevention and Research Institute of Texas (RR160088)

  • David W Taylor

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

Copyright

© 2022, McCafferty 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.

Download links

Share this article

https://doi.org/10.7554/eLife.81977

Further reading

    1. Biochemistry and Chemical Biology
    2. Microbiology and Infectious Disease
    Eva Herdering, Tristan Reif-Trauttmansdorff ... Ruth Anne Schmitz
    Research Article

    Glutamine synthetases (GS) are central enzymes essential for the nitrogen metabolism across all domains of life. Consequently, they have been extensively studied for more than half a century. Based on the ATP-dependent ammonium assimilation generating glutamine, GS expression and activity are strictly regulated in all organisms. In the methanogenic archaeon Methanosarcina mazei, it has been shown that the metabolite 2-oxoglutarate (2-OG) directly induces the GS activity. Besides, modulation of the activity by interaction with small proteins (GlnK1 and sP26) has been reported. Here, we show that the strong activation of M. mazei GS (GlnA1) by 2-OG is based on the 2-OG dependent dodecamer assembly of GlnA1 by using mass photometry (MP) and single particle cryo-electron microscopy (cryo-EM) analysis of purified strep-tagged GlnA1. The dodecamer assembly from dimers occurred without any detectable intermediate oligomeric state and was not affected in the presence of GlnK1. The 2.39 Å cryo-EM structure of the dodecameric complex in the presence of 12.5 mM 2-OG demonstrated that 2-OG is binding between two monomers. Thereby, 2-OG appears to induce the dodecameric assembly in a cooperative way. Furthermore, the active site is primed by an allosteric interaction cascade caused by 2-OG-binding towards an adaption of an open active state conformation. In the presence of additional glutamine, strong feedback inhibition of GS activity was observed. Since glutamine dependent disassembly of the dodecamer was excluded by MP, feedback inhibition most likely relies on the binding of glutamine to the catalytic site. Based on our findings, we propose that under nitrogen limitation the induction of M. mazei GS into a catalytically active dodecamer is not affected by GlnK1 and crucially depends on the presence of 2-OG.

    1. Biochemistry and Chemical Biology
    2. Structural Biology and Molecular Biophysics
    Joar Esteban Pinto Torres, Mathieu Claes ... Yann G-J Sterckx
    Research Article

    African trypanosomes are the causative agents of neglected tropical diseases affecting both humans and livestock. Disease control is highly challenging due to an increasing number of drug treatment failures. African trypanosomes are extracellular, blood-borne parasites that mainly rely on glycolysis for their energy metabolism within the mammalian host. Trypanosomal glycolytic enzymes are therefore of interest for the development of trypanocidal drugs. Here, we report the serendipitous discovery of a camelid single-domain antibody (sdAb aka Nanobody) that selectively inhibits the enzymatic activity of trypanosomatid (but not host) pyruvate kinases through an allosteric mechanism. By combining enzyme kinetics, biophysics, structural biology, and transgenic parasite survival assays, we provide a proof-of-principle that the sdAb-mediated enzyme inhibition negatively impacts parasite fitness and growth.