Optimization And Degradation Of Phosphoarginine-Bearing Substrates
Date
2025-05
Authors
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Journal ISSN
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Publisher
University of Delaware
Abstract
Members of the Clp family of ATP-dependent proteases play critical
roles in protein turnover in a wide variety of organisms. These enzymes help
maintain quality control of cellular proteins and regulate diverse cellular
processes [1]. In the globally important human pathogen Mycobacterium
tuberculosis (Mtb) the ClpC1P1P2 protease, a member of this family, has
been identified as a promising target for novel antibiotics to treat drug-resistant
Mtb infections. Like all Clp proteases, ClpC1P1P2 contains two major
functional components: a ring-shaped unfoldase (ClpC1) and a barrel-shaped
peptidase (ClpP1P2) [2]. The ClpC1 unfoldase selectively recognizes protein
substrates. However, the process of substrate recognition is poorly
understood, and few specific physiological substrates have been described.
This makes it difficult to develop effective screens for ClpC1-targeting
antibiotics. Recent studies by the Clausen group (IMP, Austria) have shown
that post-translational phosphoarginine modifications (pArg) are recognized by
the ClpC unfoldase in Bacillus subtilis (Bsu) and effectively mark substrates for
destruction by the ClpCP protease [3]. The specific kinase that catalyzes
arginine phosphorylation in Bsu is McsB [4]. A recent study by our group
showed that pArg exists in mycobacteria, although the mycobacterial arginine
kinase and phosphatase remains unknown [5]. Additionally, we have found
that the pArg binding pockets present in Bsu ClpC are conserved across
Actinobacterial ClpC1s, suggesting that pArg modifications similarly function
as markers for degradation by ClpC1P1P2 in Mtb. In this study, we aimed to
optimize the in vitro generation and purification of pArg-bearing model
substrates using a panel of McsB arginine kinases from diverse bacterial
species, and to identify the most effective kinase for substrate
phosphorylation. We found that the psychrophilic kinase from Paenibacillus
glacialis (PglMcsB) achieved phosphorylation efficiency comparable to or
exceeding that of the previously used Geobacillus stearothermophilus McsB.
Furthermore, we established a robust Strep-Tactin affinity purification protocol
to obtain highly pure phosphorylated substrates. These advances improve the
toolkit for studying ClpC1P1P2 substrate recognition and lay the groundwork
for future investigations into pArg-mediated proteolysis in mycobac