Concerted regulation of ISWI by an autoinhibitory domain and the H4 N-terminal tail

  1. Johanna Ludwigsen
  2. Sabrina Pfennig
  3. Ashish K Singh
  4. Christina Schindler
  5. Nadine Harrer
  6. Ignasi Forné
  7. Martin Zacharias
  8. Felix Mueller-Planitz  Is a corresponding author
  1. Ludwig-Maximilians-Universität München, Germany
  2. Technische Universität München, Germany

Abstract

ISWI-family nucleosome remodeling enzymes need the histone H4 N-terminal tail to mobilize nucleosomes. Here we mapped the H4-tail binding pocket of ISWI. Surprisingly the binding site was adjacent to but not overlapping with the docking site of an auto-regulatory motif, AutoN, in the N-terminal region (NTR) of ISWI, indicating that AutoN does not act as a simple pseudosubstrate as suggested previously. Rather, AutoN cooperated with a hitherto uncharacterized motif, termed AcidicN, to confer H4-tail sensitivity and discriminate between DNA and nucleosomes. A third motif in the NTR, ppHSA, was functionally required in vivo and provided structural stability by clamping the NTR to Lobe 2 of the ATPase domain. This configuration is reminiscent of Chd1 even though Chd1 contains an unrelated NTR. Our results shed light on the intricate structural and functional regulation of ISWI by the NTR and uncover surprising parallels with Chd1.

Article and author information

Author details

  1. Johanna Ludwigsen

    Biomedical Center, Ludwig-Maximilians-Universität München, Martinsried, Germany
    Competing interests
    The authors declare that no competing interests exist.
  2. Sabrina Pfennig

    Biomedical Center, Ludwig-Maximilians-Universität München, Martinsried, Germany
    Competing interests
    The authors declare that no competing interests exist.
  3. Ashish K Singh

    Biomedical Center, Ludwig-Maximilians-Universität München, Martinsried, Germany
    Competing interests
    The authors declare that no competing interests exist.
  4. Christina Schindler

    Biomedical Center, Ludwig-Maximilians-Universität München, Martinsried, Germany
    Competing interests
    The authors declare that no competing interests exist.
  5. Nadine Harrer

    Biomedical Center, Ludwig-Maximilians-Universität München, Martinsried, Germany
    Competing interests
    The authors declare that no competing interests exist.
  6. Ignasi Forné

    Biomedical Center, Ludwig-Maximilians-Universität München, Martinsried, Germany
    Competing interests
    The authors declare that no competing interests exist.
  7. Martin Zacharias

    Physics Department, Technische Universität München, Garching, Germany
    Competing interests
    The authors declare that no competing interests exist.
  8. Felix Mueller-Planitz

    Biomedical Center, Ludwig-Maximilians-Universität München, Martinsried, Germany
    For correspondence
    felix.mueller-planitz@med.uni-muenchen.de
    Competing interests
    The authors declare that no competing interests exist.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0001-8273-6473

Funding

Ernst Schering Foundation

  • Johanna Ludwigsen

Deutscher Akademischer Austauschdienst

  • Ashish K Singh

Deutsche Forschungsgemeinschaft (CIPSM)

  • Martin Zacharias

Deutsche Forschungsgemeinschaft (MU 3613/1-1 MU 3613/3-1 SFB 1064/1TP-A07)

  • Felix Mueller-Planitz

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

Reviewing Editor

  1. Jerry L Workman, Stowers Institute for Medical Research, United States

Version history

  1. Received: September 13, 2016
  2. Accepted: January 20, 2017
  3. Accepted Manuscript published: January 21, 2017 (version 1)
  4. Version of Record published: February 13, 2017 (version 2)

Copyright

© 2017, Ludwigsen 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

  • 1,552
    Page views
  • 353
    Downloads
  • 18
    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. Johanna Ludwigsen
  2. Sabrina Pfennig
  3. Ashish K Singh
  4. Christina Schindler
  5. Nadine Harrer
  6. Ignasi Forné
  7. Martin Zacharias
  8. Felix Mueller-Planitz
(2017)
Concerted regulation of ISWI by an autoinhibitory domain and the H4 N-terminal tail
eLife 6:e21477.
https://doi.org/10.7554/eLife.21477

Further reading

    1. Biochemistry and Chemical Biology
    2. Structural Biology and Molecular Biophysics
    Daniel Muñoz-Reyes, Levi J McClelland ... Maria Jose Sanchez-Barrena
    Research Article

    The Neuronal Calcium Sensor 1, an EF-hand Ca2+ binding protein, and Ric-8A coregulate synapse number and probability of neurotransmitter release. Recently, the structures of Ric-8A bound to Ga have revealed how Ric-8A phosphorylation promotes Ga recognition and activity as a chaperone and guanine nucleotide exchange factor. However, the molecular mechanism by which NCS-1 regulates Ric-8A activity and its interaction with Ga subunits is not well understood. Given the interest in the NCS-1/Ric-8A complex as a therapeutic target in nervous system disorders, it is necessary to shed light on this molecular mechanism of action at atomic level. We have reconstituted NCS-1/Ric-8A complexes to conduct a multimodal approach and determine the sequence of Ca2+ signals and phosphorylation events that promote the interaction of Ric-8A with Ga. Our data show that the binding of NCS-1 and Ga to Ric-8A are mutually exclusive. Importantly, NCS-1 induces a structural rearrangement in Ric-8A that traps the protein in a conformational state that is inaccessible to Casein Kinase II-mediated phosphorylation, demonstrating one aspect of its negative regulation of Ric-8A-mediated G-protein signaling. Functional experiments indicate a loss of Ric-8A GEF activity towards Ga when complexed with NCS-1, and restoration of nucleotide exchange activity upon increasing Ca2+ concentration. Finally, the high-resolution crystallographic data reported here define the NCS-1/Ric-8A interface and will allow the development of therapeutic synapse function regulators with improved activity and selectivity.

    1. Biochemistry and Chemical Biology
    2. Cell Biology
    Riham Ayoubi, Joel Ryan ... Carl Laflamme
    Research Advance

    Antibodies are critical reagents to detect and characterize proteins. It is commonly understood that many commercial antibodies do not recognize their intended targets, but information on the scope of the problem remains largely anecdotal, and as such, feasibility of the goal of at least one potent and specific antibody targeting each protein in a proteome cannot be assessed. Focusing on antibodies for human proteins, we have scaled a standardized characterization approach using parental and knockout cell lines (Laflamme et al., 2019) to assess the performance of 614 commercial antibodies for 65 neuroscience-related proteins. Side-by-side comparisons of all antibodies against each target, obtained from multiple commercial partners, have demonstrated that: (i) more than 50% of all antibodies failed in one or more applications, (ii) yet, ~50–75% of the protein set was covered by at least one high-performing antibody, depending on application, suggesting that coverage of human proteins by commercial antibodies is significant; and (iii) recombinant antibodies performed better than monoclonal or polyclonal antibodies. The hundreds of underperforming antibodies identified in this study were found to have been used in a large number of published articles, which should raise alarm. Encouragingly, more than half of the underperforming commercial antibodies were reassessed by the manufacturers, and many had alterations to their recommended usage or were removed from the market. This first study helps demonstrate the scale of the antibody specificity problem but also suggests an efficient strategy toward achieving coverage of the human proteome; mine the existing commercial antibody repertoire, and use the data to focus new renewable antibody generation efforts.