The ClpP N-terminus coordinates substrate access with protease active site reactivity

Energy-dependent protein degradation plays a vital role in protein homeostasis in all organisms (1). Examples of proteolytic machines include the eukaryotic 26S proteasome and the bacterial proteases, FtsH, Lon, HslUV, ClpXP, and ClpAP. In these proteolytic complexes, AAA+ ATPases (ATPases associated with various cellular activities) recognize, unfold, and translocate protein substrates into barrel-shaped compartmental proteases where the proteins are hydrolyzed and released as peptide products (2–4). Despite significant sequence divergence, the overall architecture of these complexes is conserved. For example, in ClpAP, ClpXP, HslUV, and the 26S proteasome, the protease subunits (ClpP, HslV, or the 20S peptidase) form either hexameric or heptameric ring structures that stack face to face, resulting in the sequestering of the active sites in a solvent-exposed chamber (5–8). The ATPase subunits (ClpA, ClpX, HslU, or the 19S complex) form hexameric ring-shaped structures that stack co-axially with the ring-shaped proteases, creating a continuous pore through which denatured protein substrates can be passed from the ATPase subunit into the protease compartment (9–13). Understanding the communication between the ATPase component and the protease component is a key element in understanding the mechanism of protein degradation by these machines. A number of domain interactions have been shown to participate in communication between the protease ClpP and its partner ATPases, ClpX and ClpA. The IGF/L loops of ClpA and ClpX that interact with ClpP are important in the formation of ClpA/XP complexes (14–16), with all six loops being required for complex formation (17). In addition, the N-terminal ClpP axial loops interact with the pore-2 loops of ClpX (17), and mutations and deletions in the ClpP N-terminal region abolish binding to both ClpX and ClpA (18–20). Although these previous studies revealed important interactions between ClpP and ClpX/ClpA, it remains an open question as to how the IGF/L loops and the N-terminal ClpP loops may change conformation in the course of the catalytic cycle. The sequence of the ClpP N-terminus is highly conserved across many species (18, 19); however, structural studies indicate that that this region of ClpP is flexible (5, 18–23). These observations suggest that this region may play an important dynamic role in the processing of peptide substrates. ClpP structures have been solved from five different organisms, and multiple crystal structures exist for some of these species (5, 18–23) (see Ref. (24) for a review). While the overall structure of ClpP is highly conserved among the structures solved to date, a high degree of variability exists in the conformation of the N-terminal region. In structures for which electron density is observable, the N-terminus crystallizes in one of two ways—in an “up” conformation or in a “down” conformation. In the “up” conformation, which has been observed in Escherichia coli (20, 21), Streptococcus pneumoniae (19), and human (18) enzymes, the N-terminus forms a loop in which the first ~7 residues line the axial pore while residues ~8–16 form a flexible loop extending out of the pore. The “down” conformation (observed in Escherichia coli (20)) is less well-defined. In this form, no residues are visibly protruding from the pore; electron density assigned to the first eleven residues is present in the pore, but is poorly defined. In their 2006 paper, Bewley et al. published a ClpP structure in which 6 of the N-terminal loops on a ClpP heptamer were in the “up” conformation while one was in the “down” conformation (20). They suggested that this arrangement of N-termini may facilitate a local symmetry match between ClpP and ClpX/ClpA, which may play a critical role in complex formation and/or catalysis. The flexibility of the ClpP N-terminus exhibited in multiple crystal structures, along with the recent finding that it is important in interactions with its partner ATPases, suggests that the ClpP N-terminus could gate access to the ClpP degradation chamber. Such a mechanism is utilized in the proteasome (25–29). The N-termini of the 20S core particle alpha subunits occupy the pore region of this cylindrical protease. Access to the active sites is permitted only by removal of these loops, which is facilitated by the binding of its partner ATPase, the 19S regulatory particle. A similar mechanism may be employed by ClpAP and ClpXP. When exit of peptide products through the equatorial pores of ClpP is blocked, truncation of the N-terminus allows these products to escape the ClpP complex (30), suggesting that occupancy of the N-terminus in the ClpP pore interferes with transport of peptides through the complex. Here, we describe synchrotron hydroxyl radical footprinting experiments that identify conformational changes that occur in ClpP upon binding to ClpA. This technique permits relative quantification of the solvent accessibility of protein side chains, allowing interaction sites and conformational changes to be mapped to specific areas of the protein. Synchrotron protein footprinting requires the generation of a transient concentration of hydroxyl radicals by direct irradiation of the protein solution for milliseconds with a high-flux x-ray beam. These radicals produce characteristic oxidative modifications on protein side chains (31, 32) in solvent-exposed regions of the protein; areas of the protein not exposed to solvent are protected from these stable oxidations. Protease digestion of the sample followed by liquid-chromatography-coupled mass spectrometry (LCMS) and tandem mass spectrometry (MSMS) allows identification of the oxidized regions and often the specific residue that is modified as well. Binding sites and regions involved in conformational change are then identified by differences in side chain modification rate upon addition of binding partners or small molecule ligands (33, 34). The synchrotron hydroxyl radical footprinting results show that the ClpP N-terminal residues are shielded from solvent when ClpP is not in complex with ClpA but become significantly more solvent-exposed upon binding of ClpA. To investigate the functional consequences of this conformational change, we also carried out steady-state kinetic characterization of wild-type and N-terminally truncated (19) ClpP. These kinetic studies reveal that removal of the first seven residues of ClpP increases the peptidase initial rate by 5–15 fold. These results provide the first direct evidence that the ClpP N-terminus controls the enzyme's catalytic activity. Moreover, we show here that removal of the N-terminus traps the enzyme in an acyl-enzyme bound form, indicating that an allosteric conformational change involving the N-terminus is necessary to activate the enzyme for acyl-enzyme intermediate breakdown. Together, these results support the hypothesis that motions of the ClpP N-terminus in and out of the ClpP pore regulate both substrate access to the protease active sites and the catalytic efficiency of these active sites for acyl-enzyme hydrolysis.