1. Biochemistry and Chemical Biology
  2. Structural Biology and Molecular Biophysics

A histidine pH sensor regulates activation of the Ras-specific guanine nucleotide exchange factor RasGRP1

  1. Yvonne Vercoulen
  2. Yasushi Kondo
  3. Jeffrey S Iwig
  4. Alex Janssen
  5. Katharine A White
  6. Mojtaba Amini
  7. Diane L Barber
  8. John Kuriyan  Is a corresponding author
  9. Jeroen P Roose  Is a corresponding author
  1. University of California, San Francisco, United States
  2. University of California, Berkeley, United States
  3. University Medical Center Utrecht, Netherlands
https://doi.org/10.7554/eLife.29002
Share this article
Cite this article
  1. Yvonne Vercoulen
  2. Yasushi Kondo
  3. Jeffrey S Iwig
  4. Alex Janssen
  5. Katharine A White
  6. Mojtaba Amini
  7. Diane L Barber
  8. John Kuriyan
  9. Jeroen P Roose
(2017)
A histidine pH sensor regulates activation of the Ras-specific guanine nucleotide exchange factor RasGRP1
eLife 6:e29002.
https://doi.org/10.7554/eLife.29002
  2. Download BibTeX
  3. Download .RIS

Abstract

RasGRPs are guanine nucleotide exchange factors that are specific for Ras or Rap, and are important regulators of cellular signaling. Aberrant expression or mutation of RasGRPs results in disease. An analysis of RasGRP1 SNP variants led to the conclusion that the charge of His 212 in RasGRP1 alters signaling activity and plasma membrane recruitment, indicating that His 212 is a pH sensor that alters the balance between the inactive and active forms of RasGRP1. To understand the structural basis for this effect we compared the structure of autoinhibited RasGRP1, determined previously, to that of active RasGRP4:H-Ras and RasGRP2:Rap1b complexes. The transition from the autoinhibited to the active form of RasGRP1 involves the rearrangement of an inter-domain linker that displaces inhibitory inter-domain interactions. His 212 is located at the fulcrum of these conformational changes, and structural features in its vicinity are consistent with its function as a pH-dependent switch.

Data availability

The following data sets were generated
    1. Kondo
    2. Y.
    3. Iwig
    4. J.S.
    5. Kuriyan
    6. J.
    (2017) Structure of RasGRP2 in complex with Rap1B
    Publicly available at the RCSB Protein Data Bank (Accession no: 6AXF).
    http://www.pdb.org/pdb/search/structidSearch.do?structureId=6AXF
    1. Kondo
    2. Y.
    3. Iwig
    4. J.S.
    5. Kuriyan
    6. J.
    (2017) Structure of RasGRP4 in complex with HRas
    Publicly available at the RCSB Protein Data Bank (Accession no: 6AXG).
    http://www.pdb.org/pdb/search/structidSearch.do?structureId=6AXG

Article and author information

Author details

  1. Yvonne Vercoulen

    Department of Anatomy, University of California, San Francisco, San Francisco, United States
    Competing interests
    No competing interests declared.

  2. Yasushi Kondo

    Department of Molecular and Cell Biology, University of California, Berkeley, Berkeley, United States
    Competing interests
    No competing interests declared.

  3. Jeffrey S Iwig

    Department of Molecular and Cell Biology, University of California, Berkeley, Berkeley, United States
    Competing interests
    No competing interests declared.

  4. Alex Janssen

    Department of Anatomy, University of California, San Francisco, San Francisco, United States
    Competing interests
    No competing interests declared.

  5. Katharine A White

    Department of Cell and Tissue Biology, University of California, San Francisco, San Francisco, United States
    Competing interests
    No competing interests declared.

  6. Mojtaba Amini

    Molecular Cancer Research, Center for Molecular Medicine, University Medical Center Utrecht, Utrecht, Netherlands
    Competing interests
    No competing interests declared.

  7. Diane L Barber

    Department of Cell and Tissue Biology, University of California, San Francisco, San Francisco, United States
    Competing interests
    No competing interests declared.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0001-7185-9435

  8. John Kuriyan

    Department of Molecular and Cell Biology, University of California, Berkeley, Berkeley, United States
    For correspondence
    jkuriyan@mac.com
    Competing interests
    John Kuriyan, Senior editor, eLife.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0002-4414-5477

  9. Jeroen P Roose

    Department of Anatomy, University of California, San Francisco, San Francisco, United States
    For correspondence
    jeroen.roose@ucsf.edu
    Competing interests
    No competing interests declared.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0003-4746-2811

Funding

National Institute of Allergy and Infectious Diseases

  • Jeroen P Roose

National Cancer Institute

  • Katharine A White
  • Diane L Barber

Marie Curie Cancer Care

  • Yvonne Vercoulen

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

Copyright

© 2017, Vercoulen 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

  • 3,083
    views
  • 468
    downloads
  • 35
    citations

Views, downloads and citations are aggregated across all versions of this paper published by eLife.

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. Yvonne Vercoulen
  2. Yasushi Kondo
  3. Jeffrey S Iwig
  4. Alex Janssen
  5. Katharine A White
  6. Mojtaba Amini
  7. Diane L Barber
  8. John Kuriyan
  9. Jeroen P Roose
(2017)
A histidine pH sensor regulates activation of the Ras-specific guanine nucleotide exchange factor RasGRP1
eLife 6:e29002.
https://doi.org/10.7554/eLife.29002

Share this article

https://doi.org/10.7554/eLife.29002

Categories and tags

Further reading

    1. Biochemistry and Chemical Biology

    DTX3L ubiquitin ligase ubiquitinates single-stranded nucleic acids

    Emily L Dearlove, Chatrin Chatrin ... Danny T Huang
    Research Article

    Ubiquitination typically involves covalent linking of ubiquitin (Ub) to a lysine residue on a protein substrate. Recently, new facets of this process have emerged, including Ub modification of non-proteinaceous substrates like ADP-ribose by the DELTEX E3 ligase family. Here, we show that the DELTEX family member DTX3L expands this non-proteinaceous substrate repertoire to include single-stranded DNA and RNA. Although the N-terminal region of DTX3L contains single-stranded nucleic acid binding domains and motifs, the minimal catalytically competent fragment comprises the C-terminal RING and DTC domains (RD). DTX3L-RD catalyses ubiquitination of the 3’-end of single-stranded DNA and RNA, as well as double-stranded DNA with a 3’ overhang of two or more nucleotides. This modification is reversibly cleaved by deubiquitinases. NMR and biochemical analyses reveal that the DTC domain binds single-stranded DNA and facilitates the catalysis of Ub transfer from RING-bound E2-conjugated Ub. Our study unveils the direct ubiquitination of nucleic acids by DTX3L, laying the groundwork for understanding its functional implications.

    1. Biochemistry and Chemical Biology

    Single turnover transient state kinetics reveals processive protein unfolding catalyzed by Escherichia coli ClpB

    Jaskamaljot Kaur Banwait, Liana Islam, Aaron L Lucius
    Research Article

    Escherichia coli ClpB and Saccharomyces cerevisiae Hsp104 are AAA+ motor proteins essential for proteome maintenance and thermal tolerance. ClpB and Hsp104 have been proposed to extract a polypeptide from an aggregate and processively translocate the chain through the axial channel of its hexameric ring structure. However, the mechanism of translocation and if this reaction is processive remains disputed. We reported that Hsp104 and ClpB are non-processive on unfolded model substrates. Others have reported that ClpB is able to processively translocate a mechanically unfolded polypeptide chain at rates over 240 amino acids (aa) per second. Here, we report the development of a single turnover stopped-flow fluorescence strategy that reports on processive protein unfolding catalyzed by ClpB. We show that when translocation catalyzed by ClpB is challenged by stably folded protein structure, the motor enzymatically unfolds the substrate at a rate of ~0.9 aa s−1 with a kinetic step-size of ~60 amino acids at sub-saturating [ATP]. We reconcile the apparent controversy by defining enzyme catalyzed protein unfolding and translocation as two distinct reactions with different mechanisms of action. We propose a model where slow unfolding followed by fast translocation represents an important mechanistic feature that allows the motor to rapidly translocate up to the next folded region or rapidly dissociate if no additional fold is encountered.

    1. Biochemistry and Chemical Biology
    2. Structural Biology and Molecular Biophysics

    Dynamic basis of lipopolysaccharide export by LptB2FGC

    Marina Dajka, Tobias Rath ... Benesh Joseph
    Research Article

    Lipopolysaccharides (LPS) confer resistance against harsh conditions, including antibiotics, in Gram-negative bacteria. The lipopolysaccharide transport (Lpt) complex, consisting of seven proteins (A-G), exports LPS across the cellular envelope. LptB2FG forms an ATP-binding cassette transporter that transfers LPS to LptC. How LptB2FG couples ATP binding and hydrolysis with LPS transport to LptC remains unclear. We observed the conformational heterogeneity of LptB2FG and LptB2FGC in micelles and/or proteoliposomes using pulsed dipolar electron spin resonance spectroscopy. Additionally, we monitored LPS binding and release using laser-induced liquid bead ion desorption mass spectrometry. The β-jellyroll domain of LptF stably interacts with the LptG and LptC β-jellyrolls in both the apo and vanadate-trapped states. ATP binding at the cytoplasmic side is allosterically coupled to the selective opening of the periplasmic LptF β-jellyroll domain. In LptB2FG, ATP binding closes the nucleotide binding domains, causing a collapse of the first lateral gate as observed in structures. However, the second lateral gate, which forms the putative entry site for LPS, exhibits a heterogeneous conformation. LptC binding limits the flexibility of this gate to two conformations, likely representing the helix of LptC as either released from or inserted into the transmembrane domains. Our results reveal the regulation of the LPS entry gate through the dynamic behavior of the LptC transmembrane helix, while its β-jellyroll domain is anchored in the periplasm. This, combined with long-range ATP-dependent allosteric gating of the LptF β-jellyroll domain, may ensure efficient and unidirectional transport of LPS across the periplasm.