ClpX Inhibits FtsZ Assembly in a Manner That Does Not Require Its ATP Hydrolysis-Dependent Chaperone Activity

Abstract

ClpX is a well-characterized bacterial chaperone that plays a role in many processes, including protein turnover and the remodeling of macromolecular complexes. All of these activities require ATP hydrolysisdependent, ClpX-mediated protein unfolding. Here we used site-directed mutagenesis in combination with genetics and biochemistry to establish that ClpX inhibits assembly of the conserved division protein FtsZ through a noncanonical mechanism independent of its role as an ATP-dependent chaperone.The highly conserved chaperone ClpX has been implicated in numerous cellular processes, including the turnover of regulatory proteins (6,13,19,20,32), the destruction of unfinished polypeptides (8,30,31), and the remodeling of macromolecular complexes (3-5). While ClpX-mediated proteolysis is dependent on the ClpP protease, ClpX alone is sufficient for remodeling of macromolecular complexes, such as the phage Mu transpososome (3-5). All of these functions share a requirement for ATP hydrolysis-dependent, ClpX-mediated protein unfolding.In Bacillus subtilis, ClpX modulates assembly of the conserved cytoskeletal protein FtsZ to help control the process of cell division (29). FtsZ, a homolog of the eukaryotic cytoskeletal protein tubulin, assembles into a ring structure at the nascent division site in response to an unidentified cell cycle signal. This ring serves as the foundation for assembly of the cell division apparatus (25). In previous work, we determined that ClpX inhibits FtsZ assembly and maintains the pool of subunits available for ring formation (29). Surprisingly, our biochemical data suggested that ClpX inhibits FtsZ assembly through a ClpP-independent mechanism that does not appear to require ATP hydrolysis (29).Here we employed site-directed mutagenesis in combination with genetic and biochemical analyses to establish that ClpX has two functions in vivo: an ATP hydrolysis-dependent chaperone activity, required for the unfolding of proteolytic substrates, and an ATP hydrolysis-independent activity, required for interaction with FtsZ.Generating mutations in ClpX residues required for chaperone activity. To generate mutations that render B. subtilis ClpX defective for ATP-dependent protein unfolding, we took advantage of an alignment with the well-characterized Escherichia coli ClpX protein (Fig. 1A) to engineer single alanine substitutions in B. subtilis ClpX residues that are predicted to be required for ClpX-mediated proteolysis. These substitutions included mutations in the putative Walker A [ClpX(K122A)] and Walker B [ClpX(E182A)] boxes, which in E. coli are required for ATP binding and hydrolysis, respectively, as well as a mutation in the substrate-processing pore loop [ClpX(Y150A)] (12). ATP binding is necessary for formation of the active ClpX hexamer (2, 9, 27), whereas ATP hydrolysis provides the energy required for protein unfolding and translocation (12). Mutations in the E. coli ClpX Walker A and Walker B motifs disrupt ClpX-mediated proteolysis and macromolecular complex remodeling (reviewed in...

AAA+ Chaperone ClpX Regulates Dynamics of Prokaryotic Cytoskeletal Protein FtsZ

“…4) would lead to the reduction of the freely accessible N-terminal domain of ClpX. A recent finding that a mutation into the Walker B motif (E182A) reduced the ClpX activity to inhibit FtsZ assembly in vitro (20) can also be accounted for by our proposed molecular mechanism, because the ATP hydrolysis mutant, ClpX (E182A), might be kept in a state of ATPbound hexamer. Taking into consideration that the negative effect of ADP was smaller than that of ATP and ATP␥S, the ADP-bound but not ATP-bound ClpX is in a preferred conformation for FtsZ recognition.…”

“…Recently, by the same group it has been reported that a ClpX mutant (E182A), which is defective in the ATP hydrolysis, showed a reduced ability to inhibit FtsZ polymerization in vitro, and thus it has been speculated that ATP hydrolysis is required for maximum inhibition (20). Therefore, the significance of ATP hydrolysis remains to be elucidated.…”

“…Recently, it has been reported that in Bacillus subtilis ClpX inhibits FtsZ polymerization in vivo and in vitro in an ATP-independent manner but does not degrade it (19,20). However, molecular mechanisms of regulating FtsZ monomerpolymer dynamics are still largely unknown.…”

Characterization of CrgA, a New Partner of the Mycobacterium tuberculosis Peptidoglycan Polymerization Complexes

“…MciZ is a 40-amino-acid (aa) peptide involved in the inhibition of FtsZ ring assembly following sporulation in B. subtilis (29). ClpX, a part of the ClpXP protease complex, inhibits FtsZ polymerization and possibly helps to maintain cytoplasmic pools of unpolymerized FtsZ subunits (5,20,25,44,50). Finally, the Min system spatially regulates cell division by preventing FtsZ assembly at the cell poles (27).…”

ClpXP and ClpAP control the Escherichia coli division protein ZapC by proteolysis

“…In addition to its role in the ClpXP complex, ClpX alone acts as an ATP-dependent chaperone to remodel protein substrates (Baker & Sauer, 2012). Control of FtsZ assembly mediated by ClpXP or ClpX alone has been observed in model bacteria including Bacillus, Caulobacter, Staphylococcus and Mycobacterium (Dziedzic et al, 2010;Feng et al, 2013;Haeusser et al, 2009;Weart et al, 2005;Williams et al, 2014). Notably, the precise mechanisms by which ClpX, alone or together with ClpP, influences FtsZ assembly in these bacteria are varied and are likely dependent on their specific environmental niches.…”