Versatile modes of peptide recognition by the ClpX N domain mediate alternative adaptor-binding specificities in different bacterial species

Department of Biology, Massachusetts Institute of Technology, Cambridge, Massachusetts 02139, USA.
Protein Science (Impact Factor: 2.85). 02/2010; 19(2):242-54. DOI: 10.1002/pro.306
Source: PubMed


ClpXP, an AAA+ protease, plays key roles in protein-quality control and many regulatory processes in bacteria. The N-terminal domain of the ClpX component of ClpXP is involved in recognition of many protein substrates, either directly or by binding the SspB adaptor protein, which delivers specific classes of substrates for degradation. Despite very limited sequence homology between the E. coli and C. crescentus SspB orthologs, each of these adaptors can deliver substrates to the ClpXP enzyme from the other bacterial species. We show that the ClpX N domain recognizes different sequence determinants in the ClpX-binding (XB) peptides of C. crescentus SspBalpha and E. coli SspB. The C. crescentus XB determinants span 10 residues and involve interactions with multiple side chains, whereas the E. coli XB determinants span half as many residues with only a few important side chain contacts. These results demonstrate that the N domain of ClpX functions as a highly versatile platform for peptide recognition, allowing the emergence during evolution of alternative adaptor-binding specificities. Our results also reveal highly conserved residues in the XB peptides of both E. coli SspB and C. crescentus SspBalpha that play no detectable role in ClpX-binding or substrate delivery.

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Available from: Peter Chien, Mar 28, 2014
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    • "In vitro studies of adaptor-mediated proteolysis catalysed by Clp proteases involve reactions in which the adaptor, such as SspB and ClpS, is present at equimolar or near equimolar amounts with respect to protease and substrate (Levchenko et al., 2000). In one case, SspB was present in 10-fold molar excess over ClpXP (Chowdhury et al., 2010), while in another study, ClpS was present in a ClpAPcatalysed proteolytic reaction in 0.5 molar equivalent to protease and substrate (Dougan et al., 2002). Thus, the adaptor–protease complex acts catalytically in the reaction that causes substrate degradation, but the adaptor acts stoichiometrically with respect to interaction with the protease. "
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    ABSTRACT: Proteolytic control can govern the levels of specific regulatory factors, such as Spx, a transcriptional regulator of the oxidative stress response in Gram-positive bacteria. Under oxidative stress, Spx concentration is elevated and upregulates transcription of genes that function in the stress response. When stress is alleviated, proteolysis of Spx catalysed by ClpXP reduces Spx concentration. Proteolysis is enhanced by the substrate recognition factor YjbH, which possesses a His-Cys-rich region at its N terminus. However, mutations that generate H12A, C13A, H14A, H16A and C31/34A residue substitutions in the N terminus of Bacillus subtilis YjbH (BsYjbH) do not affect functionality in Spx proteolytic control in vivo and in vitro. Because of difficulties in obtaining soluble BsYjbH, the Geobacillus thermodenitrificans yjbH gene was cloned, which yielded soluble GtYjbH protein. Despite its lack of a His-Cys-rich region, GtYjbH complements a B. subtilis yjbH null mutant, and shows high activity in vitro when combined with ClpXP and Spx in an approximately 30 : 1 (ClpXP/Spx : GtYjbH) molar ratio. In vitro interaction experiments showed that Spx and the protease-resistant Spx(DD) (in which the last two residues of Spx are replaced with two Asp residues) bind to GtYjbH, but deletion of 12 residues from the Spx C terminus (SpxΔC) significantly diminished interaction and proteolytic degradation, indicating that the C terminus of Spx is important for YjbH recognition. These experiments also showed that Spx, but not GtYjbH, interacts with ClpX. Kinetic measurements for Spx proteolysis by ClpXP in the presence and absence of GtYjbH suggest that YjbH overcomes non-productive Spx-ClpX interaction, resulting in rapid degradation.
    Microbiology 02/2012; 158(Pt 5):1268-78. DOI:10.1099/mic.0.057661-0 · 2.56 Impact Factor
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    ABSTRACT: Protein degradation is a central component of all biological processes. The proteome must constantly change in response to environmental stimuli. As a result, protein synthesis and regulated proteolysis are vital to cell survival. In Escherichia coli, the protease HslUV is one of five ATP-dependent proteases that shoulder the major burden of intracellular protein degradation. Although ample data exist for describing the structural architecture of the HslUV protease, very little is known about its mechanisms of function. I took a two-pronged approach to understand the functional principles that govern this protease. My first goal was to understand the rules of substrate recognition. To do so, I performed a variety of experiments on two model proteins: Arc repressor and [chi]cIN repressor. I found that both substrates had common requirements for HslUV degradation, suggesting a conserved mode of recognition by this protease. Mutagenesis of either substrate terminus affected binding and degradation kinetics. While degron mutations generally affect only enzyme-substrate binding properties in other bacterial proteases, the changes described here often affected the maximal rate of HslUV degradation. Moreover, specific occlusion of either the N-terminus or C-terminus of these substrates resulted in a substantial defect in degradation. A synergistic inhibitory effect was observed for the simultaneous masking of both termini. These results suggested a mechanism of tethering prior to engagement for degradation of HslUV substrates. I then sought to define the regions of HslU that were important for recognition and found that two segments, the GYVG pore loop and the intermediate (I) domain, played crucial roles. Investigation of mutants altered at these sites supported a mechanism of tethering of the substrate C-terminus to the I domain and engagement of the substrate N-terminus in the pore. I showed that degradation of an Arc substrate proceeds processively from the N-terminus towards the Cterminus, lending further support to this idea. Interestingly, I also discovered that the I domain plays a very important role in ATP hydrolysis by HslU and coordinates substrate recognition and stimulation of ATP turnover. This trait appears to be unique for HslU and is not a property of the accessory domains of other AAA+ protein unfolding machines.
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