Interactome analysis identifies MSMEI_3879 as a substrate of Mycolicibacterium smegmatis ClpC1

Abstract

The prevalence of drug resistant Mycobacterium tuberculosis infections has prompted extensive efforts to exploit new mycobacterial drug targets. ClpC1, the unfoldase component of the essential ClpC1P1P2 protease, has emerged as one particularly promising antibacterial target. However, efforts to identify and characterize ClpC1-targeting compounds are constrained by our limited knowledge of Clp protease function and regulation. To expand our understanding of ClpC1 physiology, we employed a co-immunoprecipitation and mass spectrometry workflow to identify proteins that interact with ClpC1 in Mycolicibacterium smegmatis, a relative of M. tuberculosis. We identify a diverse panel of interaction partners, many of which make co-immunoprecipitate with both the regulatory N-terminal domain and the ATPase core of ClpC1. Notably, our interactome analysis identifies MSMEI_3879, a truncated gene product unique to M. smegmatis, as a novel proteolytic substrate. Degradation of MSMEI_3879 by ClpC1P1P2 in vitro requires an exposed N-terminal sequence, reinforcing the idea that ClpC1 selectively recognizes disordered motifs. Fluorescent substrates incorporating MSMEI_3879 may be useful in screening for novel ClpC1-targeting antibiotics, to help address the challenge of M. tuberculosis drug resistance.