Edited by Wolfgang Peti Mycobacterium tuberculosis can remain dormant in the host, an ability that explains the failure of many current tuberculosis treatments. Recently, the natural products cyclomarin, ecumicin, and lassomycin have been shown to efficiently kill Mycobacterium tuberculosis persisters. Their target is the N-terminal domain of the hexameric AAA؉ ATPase ClpC1, which recognizes, unfolds, and translocates protein substrates, such as proteins containing phosphorylated arginine

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... es, to the ClpP1P2 protease for degradation. Surprisingly, these antibiotics do not inhibit ClpC1 ATPase activity, and how they cause cell death is still unclear. Here, using NMR and small-angle X-ray scattering, we demonstrate that arginine-phosphate binding to the ClpC1 N-terminal domain induces millisecond dynamics. We show that these dynamics are caused by conformational changes and do not result from unfolding or oligomerization of this domain. Cyclomarin binding to this domain specifically blocked these N-terminal dynamics. On the basis of these results, we propose a mechanism of action involving cyclomarin-induced restriction of ClpC1 dynamics, which modulates the chaperone enzymatic activity leading eventually to cell death. Tuberculosis (TB) 3 is a major public health problem with 10 million people infected and 2 million dying from this disease each year (1). The main challenge in the treatment of TB is the long duration of therapy required for a cure, as the resistance of TB results from its ability to stay dormant for long periods of time in the host. Most antibiotics require bacterial replication for their action, and this dormant state renders Mycobacterium tuberculosis (Mtb) resistant to bactericidal antibiotics. Aggravating this problem, Mtb has become increasingly resistant to existing antibiotics, and multidrug-resistant TB is now widespread (1). The proteolytic complex formed by the proteins MtbClpP1 and MtbClpP2 and their hexameric regulatory ATPases MtbClpX and MtbClpC1 is essential in mycobacteria and has emerged as an attractive target for anti-TB drug development. The Clp ATP-dependent protease complex is formed by two heptameric rings of protease subunits (MtbClpP1 and MtbClpP2) enclosing a central degradation chamber and a hexameric ATPase complex, MtbClpC1 or MtbClpX (2). The ClpC1/ClpX ATPases recognize, unfold, and translocate specific protein substrates into the MtbClpP1P2 proteolytic chamber, where degradation occurs. MtbClpC1 is a member of the class II AAAϩ family of proteins, which contains an N-terminal domain (NTD) and two distinct ATP-binding modules, D1 and D2 (Fig. 1a) . The active form of ClpC is a homohexamer, and in MtbClpC1 and Synechococcus elongatus ClpC ATP alone is essential and sufficient for efficient protein degradation in association with ClpP (3). However, Bacillus subtilis ClpC (BsClpC) requires the binding of both ATP and the adaptor protein MecA for the formation of the active hexamer. No homologous adaptor protein has been described in Mtb (4), but it remains to be tested whether MtbClpC1 can associate with a MecA-like protein. Recently, Clausen and co-workers (5) demonstrated that BsClpC specifically recognizes proteins phosphorylated on arginine residues by the arginine kinase McsB. These phosphorylation sites are often found in secondary structure elements and thus are accessible only when the protein is unfolded or misfolded. This innovative work revealed a new pathway for selective degradation of misfolded proteins in bacteria, but the structural consequences of arginine-phosphate (ArgP) binding to ClpC are unclear. Indeed, although the crystal structure of