ClpS Directs Degradation of N-Degron Substrates With Primary Destabilizing Residues in Mycolicibacterium smegmatis
Date
2024-12-03
Journal Title
Journal ISSN
Volume Title
Publisher
Molecular Microbiology
Abstract
Drug-resistant tuberculosis infections are a major threat to global public health. The essential mycobacterial ClpC1P1P2 protease has received attention as a prospective target for novel antibacterial therapeutics. However, efforts to probe its function in cells are constrained by our limited knowledge of its physiological proteolytic repertoire. Here, we interrogate the role of mycobacterial ClpS in directing N-degron pathway proteolysis by ClpC1P1P2 in Mycolicibacterium smegmatis. Binding assays demonstrate that mycobacterial ClpS binds canonical primary destabilizing residues (Leu, Phe, Tyr, Trp) with moderate affinity. N-degron binding restricts the conformational flexibility of a loop adjacent to the ClpS N-degron binding pocket and strengthens ClpS•ClpC1 binding affinity ~30-fold, providing a mechanism for cells to prioritize N-degron proteolysis when substrates are abundant. Proteolytic reporter assays in M. smegmatis confirm degradation of substrates bearing primary N-degrons, but suggest that secondary N-degrons are absent in mycobacteria. This work expands our understanding of the mycobacterial N-degron pathway and identifies ClpS as a critical component for substrate specificity, providing insights that may support the development of improved Clp protease inhibitors.
Graphical Abstract available at: https://doi.org/10.1111/mmi.15334
Many bacteria possess an N-degron pathway that links the proteolytic stability of proteins to the identity of their N-terminal residue. Here, we report that Mycolicibacterium smegmatis possesses a physiological N-degron pathway that is less complex than those described in other bacteria.
Description
This is the peer reviewed version of the following article: Presloid, C.J., Jiang, J., Kandel, P., Anderson, H.R., Beardslee, P.C., Swayne, T.M. and Schmitz, K.R. (2025), ClpS Directs Degradation of N-Degron Substrates With Primary Destabilizing Residues in Mycolicibacterium smegmatis. Mol Microbiol, 123: 16-30. https://doi.org/10.1111/mmi.15334, which has been published in final form at https://doi.org/10.1111/mmi.15334. This article may be used for non-commercial purposes in accordance with Wiley Terms and Conditions for Use of Self-Archived Versions. This article may not be enhanced, enriched or otherwise transformed into a derivative work, without express permission from Wiley or by statutory rights under applicable legislation. Copyright notices must not be removed, obscured or modified. The article must be linked to Wiley’s version of record on Wiley Online Library and any embedding, framing or otherwise making available the article or pages thereof by third parties from platforms, services and websites other than Wiley Online Library must be prohibited.
© 2024 John Wiley & Sons Ltd.
This article will be embargoed until 12/03/2025.
Keywords
mycobacteria, N-degron, proteolysis
Citation
Presloid, C.J., Jiang, J., Kandel, P., Anderson, H.R., Beardslee, P.C., Swayne, T.M. and Schmitz, K.R. (2025), ClpS Directs Degradation of N-Degron Substrates With Primary Destabilizing Residues in Mycolicibacterium smegmatis. Mol Microbiol, 123: 16-30. https://doi.org/10.1111/mmi.15334