Allosteric modulation of the Lon protease by effector binding and local charges

  1. Justyne L Ogdahl
  2. Peter Chien

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Abstract

The ATPase Associated with diverse cellular Activities (AAA+) family of proteases play crucial roles in cellular proteolysis and stress responses. Like other AAA+ proteases, the Lon protease is known to be allosterically regulated by nucleotide and substrate binding. Although it was originally classified as a DNA binding protein, the impact of DNA binding on Lon activity is unclear. In this study, we characterize the regulation of Lon by single-stranded DNA (ssDNA) binding and serendipitously identify general activation strategies for Lon. Upon binding to ssDNA, Lon’s ATP hydrolysis rate increases due to improved nucleotide binding, leading to enhanced degradation of protein substrates, including physiologically important targets. We demonstrate that mutations in basic residues that are crucial for Lon’s DNA binding not only reduces ssDNA binding but result in charge-specific consequences on Lon activity. Introducing negative charge at these sites induces activation akin to that induced by ssDNA binding, whereas neutralizing the charge reduces Lon’s activity. Based on single molecule measurements we find that this change in activity is correlated with changes in Lon oligomerization. Our study provides insights into the complex regulation of the Lon protease driven by electrostatic contributions from either DNA binding or mutations.

Highlights

  • ssDNA binding allosterically activates Lon ATP hydrolysis

  • Negative charge at DNA binding site is sufficient for Lon activation

  • Neutralization of charge at DNA binding site inhibits Lon ATP hydrolysis

  • Lon activity is linked to formation of stable Lon hexamers

Significance

The energy-dependent protease Lon is integral in both eukaryotic and prokaryotic physiology, contributing to protein quality control, stress management, developmental regulation, and pathogenicity. The ability to precisely regulate protein levels through targeted degradation underscores a need for tunability. We find that single-stranded DNA (ssDNA) acts as an allosteric regulator of Lon, leading to enhanced enzymatic activity. Mutations in basic residues crucial for DNA binding were found to affect Lon activity in a charge-specific manner highlighting the importance of electrostatic interactions regulating Lon’s function. Changes in Lon activity due to ssDNA binding or mutations were correlated with its oligomerization state. Our findings provide insights into the activation strategies of Lon, emphasizing the role of electrostatic contribution that modulate nucleotide affinity, oligomerization and proteolysis to advance our understanding of the complex regulatory mechanisms of the Lon protease.

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  1. PREreview

    This Zenodo record is a permanently preserved version of a PREreview. You can view the complete PREreview at https://prereview.org/reviews/14451115.

    Summary:

    This paper investigates the role of ssDNA binding in modulating Lon protease activity and explores its biochemical and structural consequences. It characterizes ssDNA binding and demonstrates how mutations that alter DNA binding impact biochemical activity without significantly affecting Lon oligomerization. Additionally, the study highlights alternative pathways through which Lon protease retains peptidase activity in the presence of ATP. Overall, the paper offers valuable insights into how DNA binding and electrostatic changes regulate Lon protease function. However, improvements could be made in the flow of paper, increasing sample sizes for certain experiments, and revising the title to better reflect the study's findings.

    Major Concerns: 1. We believe the paper can be better communicated with a different title, emphasizing Lon's impact with ssDNA.We suggest renaming it to "Understanding ssDNA Binding on the Activation and Regulation of Lon Protease Activity". We emphasize removing the "allosteric modulation" portion, as it does not consistently reflect a central theme of the findings.

    2. In the results section, the discussion in lines 91-97 accompanying Figure 1 reads more like a discussion. To improve clarity, we suggest removing sentences from 'with reports' (line 94) to 'protein degradation (24)' (line 96) and instead providing a concise summary of prior literature before introducing the Figure 1 results. 

    3. We strongly recommend moving the structural representation from Figure 7A to Figure 2. The inclusion of this structure in Figure 2 would provide a critical foundation for understanding the binding sites and the role of oligomerization in protease activity, which is explored in the experiments that follow. 4. For presentation purposes, the gels in Figures 3 and Supplemental Figures 1 & 2 could use better contrast or rerunning, as the bands are faint. In Figure 3C, we suggest including more replicates for all conditions to address variability in the Lon4A data or conducting statistical analysis to demonstrate that the variation is not significant. 

    5.  In Supplemental Figure 1B, sample sizes vary across experimental conditions despite the main text emphasizing consistency. We recommend ensuring uniform sample sizes.

    6. In Figure 2B, 2C, 3D, and 4, it is unclear whether the points in panels A and B are log-transformed. If so, this should be noted on the x-axis. 

    7. In  Figure 4D, the skewed fit of the lines for Lon and Lon4E raises concerns about data reliability. We recommend either reanalyzing the data or discussing how this skew impacts the interpretation of ATP hydrolysis results.

    8. In Figure 5, the oligomer cartoons on the graphs appear crowded and confusing. Consider using lightly shaded background sections correlating to oligomer sizes or an alternative representation to differentiate peaks.

    9. Figure 3 introduces the concept of allosteric modulation by ssDNA, but it doesn't emphasize this idea clearly. We suggest either making this the focus of Figure 4 or move it to Figure 4, which investigates peptide hydrolysis and activity. We believe Figure 4 would fit better after fully explaining allosteric modulation.

    Minor Concerns: 1. Lines 124-125 discussing the hexamer orientation do not directly relate to the experiments conducted, and we suggest they can be omitted.

    2. . In the results section for Figure 1, the full protein names for the abbreviations "DnaA," "SciP," and "Ccrm" should be included to aid reader comprehension.

    3.The terms "LMW" and "HMW," which are introduced in line 130, should be explained at that time.

    4. In Figure 2A, there is an inconsistency in the molecular weight for the high molecular weight peak. It is marked as 582 kDa in the figure but listed as 588 kDa in the results text.

    5. For the Figure 2 caption, specifically in line 560, there is a missing closing parenthesis.

    6. We suggest including a brief introduction to Michaelis-Menten (MM) kinetics and elaborating on the measured MM parameters would also help readers better understand the enzymatic differences observed between the wildtype and mutant Lon constructs in Table 1.

    7. In Supporting Information Figure 2, the sentence on line 662 that reads "contain 2 G4 DNA sequences" should be revised to "containing two G4 DNA sequences," while maintaining the numeric format (2xG4). 

    8. The word "casein" has inconsistent capitalization throughout the text; please revise. 

    9. In Figure 5B, the light blue shaded region is not explained in the caption or the results text; a brief explanation should be included, either in the caption or in lines 613-615, to clarify its significance. 

    Competing interests

    The authors declare that they have no competing interests.

  2. Version published to 10.1101/2024.09.06.611642v1 on bioRxiv