Phosphoarginine modulates oligomerization and repressor activity of mycobacterial ClpC2

Abstract

Phosphoarginine (pArg) modifications direct proteins for proteolytic destruction by ClpC1P1P2, an essential mycobacterial protease that has emerged as a promising antibacterial drug target against Mycobacterium tuberculosis. The broader regulatory landscape surrounding pArg is poorly understood. Here, we establish a mechanistic connection between pArg binding and the activity of ClpC2, a non-proteolytic transcriptional repressor with homology to the ClpC1 N-terminal domain. Biophysical studies reveal that ClpC2 forms concentration-dependent higher-order oligomers that bind cooperatively to operator sequences in the clpC2 promoter. A high-resolution crystal structure of the Streptomyces thermoviolaceus ClpC2 C-terminal domain reveals a conserved dimerization interface mediated by a C-terminal helix, which is sterically disrupted by pArg binding. Consequently, we find that binding of pArg, as well as some ClpC1-targeting antibiotics, disrupts ClpC2 oligomerization, dissociates ClpC2 from its operator DNA, and relieves transcriptional repression in vitro. Moreover, comparative analysis of clpC2 promoters with single versus dual operator sites predicts differences in regulatory sensitivity across mycobacterial species. Together, these findings establish ClpC2 as a pArg-responsive sensor capable of mechanistically linking elevated pArg levels to downstream transcriptional regulation.