The structure of the endogenous ESX-3 secretion system

  1. Nicole Poweleit
  2. Nadine Czudnochowski
  3. Rachel Nakagawa
  4. Donovan Trinidad
  5. Kenan C Murphy
  6. Christopher M Sassetti
  7. Oren S Rosenberg  Is a corresponding author
  1. University of California, San Francisco, United States
  2. University of Massachusetts Medical School, United States

Abstract

The ESX (or Type VII) secretion systems are protein export systems in mycobacteria and many Gram-positive bacteria that mediate a broad range of functions including virulence, conjugation, and metabolic regulation. These systems translocate folded dimers of WXG100-superfamily protein substrates across the cytoplasmic membrane. We report the cryo-electron microscopy structure of an ESX-3 system, purified using an epitope tag inserted with recombineering into the chromosome of the model organism Mycobacterium smegmatis. The structure reveals a stacked architecture that extends above and below the inner membrane of the bacterium. The ESX-3 protomer complex is assembled from a single copy of the EccB3, EccC3, and EccE3 and two copies of the EccD3 protein. In the structure, the protomers form a stable dimer that is consistent with assembly into a larger oligomer. The ESX-3 structure provides a framework for further study of these important bacterial transporters.

Data availability

The map files have been deposited at the EMDB with code 20820. The entry is online at https://www.ebi.ac.uk/pdbe/entry/emdb/EMD-20820.The model has been deposited at the PDB with the code 6UMM. It is online at http://www.rcsb.org/structure/6UMM

The following data sets were generated

Article and author information

Author details

  1. Nicole Poweleit

    Department of Medicine, University of California, San Francisco, San Francisco, United States
    Competing interests
    The authors declare that no competing interests exist.
  2. Nadine Czudnochowski

    Department of Medicine, University of California, San Francisco, San Francisco, United States
    Competing interests
    The authors declare that no competing interests exist.
  3. Rachel Nakagawa

    Department of Medicine, University of California, San Francisco, San Francisco, United States
    Competing interests
    The authors declare that no competing interests exist.
  4. Donovan Trinidad

    Department of Medicine, University of California, San Francisco, San Francisco, United States
    Competing interests
    The authors declare that no competing interests exist.
  5. Kenan C Murphy

    Department of Microbiology and Physiological Systems, University of Massachusetts Medical School, Worcester, United States
    Competing interests
    The authors declare that no competing interests exist.
  6. Christopher M Sassetti

    Department of Microbiology and Physiological Systems, University of Massachusetts Medical School, Worcester, United States
    Competing interests
    The authors declare that no competing interests exist.
  7. Oren S Rosenberg

    Department of Medicine, University of California, San Francisco, San Francisco, United States
    For correspondence
    oren.rosenberg@ucsf.edu
    Competing interests
    The authors declare that no competing interests exist.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0002-5736-4388

Funding

National Institutes of Health (1RO1AI128214)

  • Oren S Rosenberg

National Institutes of Health (1U19AI135990-01)

  • Oren S Rosenberg

National Institutes of Health (P01AI095208)

  • Oren S Rosenberg

National Institutes of Health (5T32AI060537)

  • Nicole Poweleit

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

Reviewing Editor

  1. Edward H Egelman, University of Virginia, United States

Version history

  1. Received: October 23, 2019
  2. Accepted: December 30, 2019
  3. Accepted Manuscript published: December 30, 2019 (version 1)
  4. Version of Record published: January 28, 2020 (version 2)

Copyright

© 2019, Poweleit 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

  • 3,763
    Page views
  • 498
    Downloads
  • 41
    Citations

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

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. Nicole Poweleit
  2. Nadine Czudnochowski
  3. Rachel Nakagawa
  4. Donovan Trinidad
  5. Kenan C Murphy
  6. Christopher M Sassetti
  7. Oren S Rosenberg
(2019)
The structure of the endogenous ESX-3 secretion system
eLife 8:e52983.
https://doi.org/10.7554/eLife.52983

Further reading

    1. Plant Biology
    2. Structural Biology and Molecular Biophysics
    Jiyu Xin, Yang Shi ... Xiaoling Xu
    Research Article

    Carotenoid (Car) pigments perform central roles in photosynthesis-related light harvesting (LH), photoprotection, and assembly of functional pigment-protein complexes. However, the relationships between Car depletion in the LH, assembly of the prokaryotic reaction center (RC)-LH complex, and quinone exchange are not fully understood. Here, we analyzed native RC-LH (nRC-LH) and Car-depleted RC-LH (dRC-LH) complexes in Roseiflexus castenholzii, a chlorosome-less filamentous anoxygenic phototroph that forms the deepest branch of photosynthetic bacteria. Newly identified exterior Cars functioned with the bacteriochlorophyll B800 to block the proposed quinone channel between LHαβ subunits in the nRC-LH, forming a sealed LH ring that was disrupted by transmembrane helices from cytochrome c and subunit X to allow quinone shuttling. dRC-LH lacked subunit X, leading to an exposed LH ring with a larger opening, which together accelerated the quinone exchange rate. We also assigned amino acid sequences of subunit X and two hypothetical proteins Y and Z that functioned in forming the quinone channel and stabilizing the RC-LH interactions. This study reveals the structural basis by which Cars assembly regulates the architecture and quinone exchange of bacterial RC-LH complexes. These findings mark an important step forward in understanding the evolution and diversity of prokaryotic photosynthetic apparatus.

    1. Neuroscience
    2. Structural Biology and Molecular Biophysics
    Ashton J Curtis, Jian Zhu ... Matthew G Gold
    Research Article Updated

    Ca2+/calmodulin-dependent protein kinase II (CaMKII) is essential for long-term potentiation (LTP) of excitatory synapses that is linked to learning and memory. In this study, we focused on understanding how interactions between CaMKIIα and the actin-crosslinking protein α-actinin-2 underlie long-lasting changes in dendritic spine architecture. We found that association of the two proteins was unexpectedly elevated within 2 minutes of NMDA receptor stimulation that triggers structural LTP in primary hippocampal neurons. Furthermore, disruption of interactions between the two proteins prevented the accumulation of enlarged mushroom-type dendritic spines following NMDA receptor activation. α-Actinin-2 binds to the regulatory segment of CaMKII. Calorimetry experiments, and a crystal structure of α-actinin-2 EF hands 3 and 4 in complex with the CaMKII regulatory segment, indicate that the regulatory segment of autoinhibited CaMKII is not fully accessible to α-actinin-2. Pull-down experiments show that occupation of the CaMKII substrate-binding groove by GluN2B markedly increases α-actinin-2 access to the CaMKII regulatory segment. Furthermore, in situ labelling experiments are consistent with the notion that recruitment of CaMKII to NMDA receptors contributes to elevated interactions between the kinase and α-actinin-2 during structural LTP. Overall, our study provides new mechanistic insight into the molecular basis of structural LTP and reveals an added layer of sophistication to the function of CaMKII.