Effects of local protein stability and the geometric position of the substrate degradation tag on the efficiency of ClpXP denaturation and degradation
Introduction
AAA+ ATPases transduce the energy of nucleotide-triphosphate binding and hydrolysis into cycles of conformational changes that can be used to denature proteins, solubilize aggregates, power protein secretion, and dismantle oligomeric structures composed of proteins and/or nucleic acids (Langer, 2000; Neuwald et al., 1999; Ogura and Wilkinson, 2001; Vale, 2000). One important biological function of AAA+ ATPases is to serve as essential components of compartmentalized proteases such as the eukaryotic proteasome or the bacterial ClpXP, ClpAP, Lon, HslUV, and FtsH proteases (Hoskins et al., 2001; Langer, 2000). In these proteases, the AAA+ ATPase recognizes a native protein substrate by binding to an exposed degradation tag, denatures the substrate by a process that is poorly understood, and then translocates the unfolded polypeptide into a sequestered chamber where it is degraded to small peptide fragments (Fig. 1).
We have been studying the ClpXP protease from Escherichia coli. In this AAA+ protease, ClpX forms a hexameric ring ATPase which stacks coaxially on one or both ends of the double-ring ClpP14 peptidase (Grimaud et al., 1998; Ortega et al., 2002; Wang et al., 1997). ClpX recognizes target proteins bearing any of several different classes of degradation signals, generally consisting of specific N- or C-terminal peptide sequences (Flynn et al., 2003; Gonciarz-Swiatek et al., 1999; Gottesman et al., 1998). One hallmark of these degradation signals or tags is their ability to target almost any protein for degradation. For example, the C-terminal ssrA tag makes numerous unrelated proteins susceptible to degradation by ClpXP (Burton et al., 2001; Gottesman et al., 1998; Keiler et al., 1996; Kenniston et al., 2003; Kim et al., 2000; Lee et al., 2001; Singh et al., 2000). This 11-residue degradation peptide also serves a general protease targeting function in cells, where it is appended by a cotranslational process to nascent chains on stalled bacterial ribosomes, thereby allowing efficient intracellular degradation of these ssrA-tagged proteins (Keiler et al., 1996).
The ability of the ssrA tag to target substrate proteins to ClpXP can be readily understood, as ClpX binds specifically to this peptide sequence (Flynn et al., 2001; Gottesman et al., 1998; Wah et al., 2002). It is less clear, however, how ClpX unfolds the attached native protein and how the stability of this protein influences its degradation. Increasing protein stability slows ClpXP degradation of ssrA-tagged proteins dramatically in some cases but has little effect in other cases (Burton et al., 2001; Kenniston et al., 2003; Lee et al., 2001). An attractive model is that ClpX unfolds proteins by pulling-on or applying mechanical force to the degradation tag, causing initial denaturation of protein structure adjacent to the degradation tag of the substrate (Lee et al., 2001; Matouschek, 2003). This model predicts that the stability of structural elements near the degradation tag will be more important than global stability in determining ClpXP degradation rates. Recent studies with an ssrA-tagged I27 domain of human titin lend support to this “local stability” model, as variants with destabilizing mutations in a β-sheet proximal to the ssrA tag were degraded faster by ClpXP than the wild-type protein or a variant with a destabilizing mutation in a β-sheet distant from the ssrA tag (Kenniston et al., 2003).
To probe the relationship between protein stability and ClpXP degradation more extensively, we created a model substrate by appending an ssrA tag to RNase-H* from Thermus thermophilus (Hollien and Marqusee, 1999a, Hollien and Marqusee, 1999b). For the experiments reported here, RNase-H* has several important properties. First, this thermophilic enzyme is remarkably stable, with a global free energy of unfolding of roughly 12 kcal/mol (Hollien and Marqusee, 1999b). Second, both T. thermophilus RNase-H* and a mesophilic E. coli counterpart have been characterized extensively by native-state hydrogen-exchange experiments (Chamberlain et al., 1996; Hollien and Marqusee, 1999a). Hence, the stabilities of specific backbone regions to local denaturation are known for these proteins. Third, by introducing Asp for Leu substitutions into the hydrophobic core, we were able to construct ssrA-tagged RNase-H* mutants with dramatically reduced native stabilities and a double mutant that was denatured under physiological conditions. These variants allowed us to test the importance of global thermodynamic stability in ClpXP degradation, and to assess the contributions of substrate denaturation and translocation to rates of degradation and ATP consumption. Finally, because RNase-H* is cysteine free, we were able to introduce surface cysteines by mutagenesis and to attach ssrA tags to these side chains by chemical linkage. Experiments with these RNase-H* substrates and a set of Arc substrates with side-chain linked ssrA tags have allowed us to probe the relationship between susceptibility to ClpXP degradation and the position of the degradation tag relative to the protein structure.
Section snippets
Protein production and modification
Genes expressing RNase-H* variants were constructed by PCR mutagenesis of plasmid pJH109 which encodes a cysteine-free variant of T. thermophilus RNase-H subcloned into a pAED4 vector (Hollien and Marqusee, 1999b). One set of RNase-H*-ssrA constructs had an N-terminal His6 tag and a C-terminal ssrA tag (AANDENYALAA), where the first residue of the degradation tag was also the C-terminal residue (Ala166) of the wild-type RNase-H* sequence. The His6 tag was omitted in another RNase-H*-ssrA
Degradation of RNase-H*-ssrA protein by ClpXP
We constructed and purified a variant of RNase-H* from T. thermophilus with a His6 tag at its N-terminus and an ssrA tag at its C-terminus. 35S-labeled RNase-H*-ssrA was efficiently degraded by ClpXP (see inset to Fig. 2). No degradation was observed if ClpX, ClpP, or ATP were omitted from the reaction. To determine kinetic parameters for ClpXP proteolysis, initial rates of degradation were measured at a set of RNase-H*-ssrA substrate concentrations (Fig. 2). These data fit well to a simple
Discussion
Protein denaturation is a prerequisite for ClpXP degradation because native proteins are excluded from the proteolytic chamber of ClpP by small entry portals (Wang et al., 1997). Nevertheless, for RNase-H*-ssrA and its variants, we find that global thermodynamic stability is uncorrelated with the rate of ClpXP denaturation or degradation. For example, although the L78D and L112D mutations reduce the stability of wild-type RNase-H*-ssrA by 7 and 9 kcal/mol, respectively, the L78D mutant is
Acknowledgements
We thank Susan Marqusee for the plasmid vector encoding T. thermophilus RNase-H*, Tony Anderson for purified Arc-SC32-st11 protein, and the MIT Biopolymers Lab for synthetic peptides and mass spectrometry. Supported by NIH Grant AI-15706. T.A.B. is an employee of the Howard Hughes Medical Institute.
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