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

Interactions between a subset of substrate side chains and AAA+ motor pore loops determine grip during protein unfolding

  1. Tristan A Bell  Is a corresponding author
  2. Tania A Baker
  3. Robert T Sauer  Is a corresponding author
  1. Massachusetts Institute of Technology, United States
Research Article
  • Cited 2
  • Views 1,699
  • Annotations
Cite this article as: eLife 2019;8:e46808 doi: 10.7554/eLife.46808

Abstract

Most AAA+ remodeling motors denature proteins by pulling on the peptide termini of folded substrates, but it is not well-understood how motors produce grip when resisting a folded domain. Here, at single amino-acid resolution, we identify the determinants of grip by measuring how substrate tail sequences alter the unfolding activity of the unfoldase-protease ClpXP. The seven amino acids abutting a stable substrate domain are key, with residues 2-6 forming a core that contributes most significantly to grip. ClpX grips large hydrophobic and aromatic side chains strongly and small, polar, or charged side chains weakly. Multiple side chains interact with pore loops synergistically to strengthen grip. In combination with recent structures, our results support a mechanism in which unfolding grip is primarily mediated by non-specific van der Waal's interactions between core side chains of the substrate tail and a subset of YVG loops at the top of the ClpX axial pore.

Article and author information

Author details

  1. Tristan A Bell

    Department of Biology, Massachusetts Institute of Technology, Cambridge, United States
    For correspondence
    tribell@mit.edu
    Competing interests
    The authors declare that no competing interests exist.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0002-3668-8412
  2. Tania A Baker

    Department of Biology, Massachusetts Institute of Technology, Cambridge, United States
    Competing interests
    The authors declare that no competing interests exist.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0002-0737-3411
  3. Robert T Sauer

    Department of Biology, Massachusetts Institute of Technology, Cambridge, United States
    For correspondence
    bobsauer@MIT.EDU
    Competing interests
    The authors declare that no competing interests exist.

Funding

National Institutes of Health (GM-101988)

  • Robert T Sauer

National Institutes of Health (5T32GM-007287)

  • Tristan A Bell

Howard Hughes Medical Institute

  • Tania A Baker

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

Reviewing Editor

  1. Axel T Brunger, Stanford University, United States

Publication history

  1. Received: March 13, 2019
  2. Accepted: June 27, 2019
  3. Accepted Manuscript published: June 28, 2019 (version 1)
  4. Version of Record published: August 2, 2019 (version 2)

Copyright

© 2019, Bell 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

  • 1,699
    Page views
  • 284
    Downloads
  • 2
    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)

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

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

Further reading

    1. Biochemistry and Chemical Biology
    Rajaa Boujemaa-Paterski et al.
    Research Article Updated

    Vinculin plays a fundamental role in integrin-mediated cell adhesion. Activated by talin, it interacts with diverse adhesome components, enabling mechanical coupling between the actin cytoskeleton and the extracellular matrix. Here we studied the interactions of activated full-length vinculin with actin and the way it regulates the organization and dynamics of the Arp2/3 complex-mediated branched actin network. Through a combination of surface patterning and light microscopy experiments we show that vinculin can bundle dendritic actin networks through rapid binding and filament crosslinking. We show that vinculin promotes stable but flexible actin bundles having a mixed-polarity organization, as confirmed by cryo-electron tomography. Adhesion-like synthetic design of vinculin activation by surface-bound talin revealed that clustered vinculin can initiate and immobilize bundles from mobile Arp2/3-branched networks. Our results provide a molecular basis for coordinate actin bundle formation at nascent adhesions.

    1. Biochemistry and Chemical Biology
    2. Microbiology and Infectious Disease
    CJ Cambier et al.
    Research Article

    Several virulence lipids populate the outer cell wall of pathogenic mycobacteria (Jackson, 2014). Phthiocerol dimycocerosate (PDIM), one of the most abundant outer membrane lipids (Anderson, 1929), plays important roles in both defending against host antimicrobial programs (Camacho et al., 2001; Cox et al., 1999; Murry et al., 2009) and in evading these programs altogether (Cambier et al., 2014a; Rousseau et al., 2004). Immediately following infection, mycobacteria rely on PDIM to evade Myd88-dependent recruitment of microbicidal monocytes which can clear infection (Cambier et al., 2014b). To circumvent the limitations in using genetics to understand virulence lipids, we developed a chemical approach to track PDIM during Mycobacterium marinum infection of zebrafish. We found that PDIM's methyl-branched lipid tails enabled it to spread into host epithelial membranes to prevent immune activation. Additionally, PDIM's affinity for cholesterol promoted this phenotype; treatment of zebrafish with statins, cholesterol synthesis inhibitors, decreased spreading and provided protection from infection. This work establishes that interactions between host and pathogen lipids influence mycobacterial infectivity and suggests the use of statins as tuberculosis preventive therapy by inhibiting PDIM spread.