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

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.

Data availability

All data generated during this study are included in the manuscript and supporting files as Tables 1 and 2 and Figure 2 - figure supplement 3.

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

  • 2,143
    Page views
  • 329
    Downloads
  • 9
    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. Tristan A Bell
  2. Tania A Baker
  3. Robert T Sauer
(2019)
Interactions between a subset of substrate side chains and AAA+ motor pore loops determine grip during protein unfolding
eLife 8:e46808.
https://doi.org/10.7554/eLife.46808

Further reading

    1. Biochemistry and Chemical Biology
    Meiling Wu, Anda Zhao ... Dongyun Shi
    Research Article

    Antioxidant intervention is considered to inhibit reactive oxygen species (ROS) and alleviates hyperglycemia. Paradoxically, moderate exercise can produce ROS to improve diabetes. The exact redox mechanism of these two different approaches remains largely unclear. Here, by comparing exercise and antioxidants intervention on type 2 diabetic rats, we found moderate exercise upregulated compensatory antioxidant capability and reached a higher level of redox balance in the liver. In contrast, antioxidant intervention achieved a low-level redox balance by inhibiting oxidative stress. Both of these two interventions could promote glucose catabolism and inhibit gluconeogenesis through activation of hepatic AMPK signaling, therefore ameliorating diabetes. During exercise, different levels of ROS generated by exercise have differential regulations on the activity and expression of hepatic AMPK. Moderate exercise-derived ROS promoted hepatic AMPK glutathionylation activation. However, excessive exercise increased oxidative damage and inhibited the activity and expression of AMPK. Overall, our results illustrate that both exercise and antioxidant intervention improve blood glucose in diabetes by promoting redox balance, despite different levels of redox balance. These results indicate that the AMPK signaling activation, combined with oxidative damage markers, could act as a sensitive biomarker, reflecting the threshold of redox balance defining effective treatment in diabetes. These findings provide theoretical evidence for the precise treatment of diabetes by antioxidants and exercise.

    1. Biochemistry and Chemical Biology
    2. Cell Biology
    Edmundo G Vides, Ayan Adhikari ... Suzanne R Pfeffer
    Research Advance

    Activating mutations in the Leucine Rich Repeat Kinase 2 (LRRK2) cause Parkinson's disease and previously we showed that activated LRRK2 phosphorylates a subset of Rab GTPases (Steger et al., 2017). Moreover, Golgi-associated Rab29 can recruit LRRK2 to the surface of the Golgi and activate it there for both auto- and Rab substrate phosphorylation. Here we define the precise Rab29 binding region of the LRRK2 Armadillo domain between residues 360-450 and show that this domain, termed 'Site #1', can also bind additional LRRK2 substrates, Rab8A and Rab10. Moreover, we identify a distinct, N-terminal, higher affinity interaction interface between LRRK2 phosphorylated Rab8 and Rab10 termed 'Site #2', that can retain LRRK2 on membranes in cells to catalyze multiple, subsequent phosphorylation events. Kinase inhibitor washout experiments demonstrate that rapid recovery of kinase activity in cells depends on the ability of LRRK2 to associate with phosphorylated Rab proteins, and phosphorylated Rab8A stimulates LRRK2 phosphorylation of Rab10 in vitro. Reconstitution of purified LRRK2 recruitment onto planar lipid bilayers decorated with Rab10 protein demonstrates cooperative association of only active LRRK2 with phospho-Rab10-containing membrane surfaces. These experiments reveal a feed-forward pathway that provides spatial control and membrane activation of LRRK2 kinase activity.