clpP:Gene Product(s

ClpP is a highly conserved serine protease present throughout bacteria and eukaryota, ; it is absent in archaea, mollicutes, and some fungi . ClpP was first identified in Escherichia coli , and more than 400 studies have been published on it since its discovery. The X-ray structure of ClpP has been solved from several different organisms. All the structures show similar features: the protease is comprised of 14 subunits arranged into two heptameric rings forming a cylindrical-like structure which encloses a large chamber containing the protease active sites. Entrance into the chamber occurs through axial pores in the cylinder. ClpP typically forms complexes with AAA+ (ATPases Associated with various cellular Activities) chaperones that denature substrates and then translocate them through the axial pores into the proteolytic chamber of the protease for degradation. ClpP degrades proteins into peptides of about 7 to 8 residues which are subsequently released from the chamber. It is one of the five major proteases in E. coli which includes ClpXP, ClpAP, HslUV, FtSH and Lon protease. Proteases play an essential role in protein quality control by removing short-lived regulatory proteins, as well as proteins that are misfolded and damaged, thus maintaining cellular homeostasis. A significant proportion of protein degradation in the cell is carried out by oligomeric, cylindrical, self-compartmentalized, energy-dependent proteases. ClpP (Caseinolytic Protease) is a representative member of these cylindrical proteases.

ClpP is found in all bacteria sequenced to date except for Mollicutes . The majority of bacteria contain only one copy of ClpP, although there are exceptions such as for actinobacteria, chlamydiae, cyanobacteria, and others which have multiple isoforms of the protease. In addition to ClpP, cyanobacteria contain a ClpP paralog that does not have all 3 residues of the Ser-His-Asp catalytic triad. This inactive version of ClpP is called ClpR and is mostly present in plants in addition to cyanobacteria. For example, cyanobacteria Synechocystis PCC6803 contains three copies of ClpP and one copy of ClpR .

ClpP was first identified and mostly studied in Escherichia coli, which contains only one copy of the protease. E. coli ClpP consists of 207 amino acids including an N-terminal prosequence which acts as a regulatory peptide and undergoes autocatalytic cleavage during folding to yield a mature ClpP of 193 residues . In E. coli, ClpP forms complexes with AAA+ chaperones, ClpX and ClpA, which belong to the Clp/Hsp100 family. ClpA is an 83 kDa ATPase that contains two AAA+ domains, while ClpX contains only one AAA+ domain (46 kDa), which is homologous to the second AAA+ domain of ClpA. ClpA and ClpX are hexameric chaperones that can stack onto one or both ends of ClpP to form ClpAP or ClpXP holoenzyme complexes. Substrates are recognized, unfolded by ClpX or ClpA, and then threaded into the ClpP proteolytic chamber through the narrow axial pores for degradation. Only the unfolding and threading by the chaperones require ATP binding and hydrolysis, while proteolysis by ClpP does not require nucleotide hydrolysis. The mechanism by which ClpX or ClpA binds to and unfolds substrates is poorly understood. Other bacteria contain ClpA paralogs, such as ClpC, ClpE, and ClpL .

The most general substrates for the E. coli ClpXP or ClpAP system are those with an SsrA C-terminal tag . The SsrA tag consists of 11 hydrophobic residues that are added by a tmRNA, also called SsrA or 10Sa RNA, to the C-terminus of nascent chains whose translation is stalled on the ribosome, targeting them for degradation . SsrA tagging can be thought of as the prokaryotic equivalent to eukaryotic ubiquitination.

Without ATPase components, only small peptides can enter the ClpP chamber ; hence, ClpP on its own cannot degrade folded proteins. Recently, however, a group at Bayer described the discovery of acyldepsipeptides (ADEPs) that, when added to ClpP, allow the protease to degrade folded native proteins in the absence of its cognate chaperones . The drug was initially found to rescue mice challenged with lethal infections of Enterococcus faecalis and Staphylococcus aureus, and the target for the drug was then identified to be ClpP. ClpP is activated by ADEPs to degrade, or at least to cleave, folded proteins in the absence of the cognate Clp ATPases. The consequences of the action of the drug in vivo, is that the ADEPs cause unregulated proteolysis by ClpP, subsequently, triggering cell death in gram-positive bacteria. Gram-negative bacteria, on the other hand, were found to be able to survive drug treatment because they have efficient efflux pumps to remove the drug from the cell, but deletion of those pumps or the use of permeabilizing agents made gram-negative bacteria vulnerable to ADEP treatment as well . The mechanism of how ADEPs activate ClpP in the absence of its cognate chaperones is currently unknown. The discovery of ADEPs may lead to development of a novel class of antimicrobial agents.

Understanding how the ClpP system functions has direct implications on understanding the mechanism of function of other more complex cylindrical proteases such as the proteasome. ClpP shares many similarities with the proteasome both functionally and structurally. The arrangement of subunits in ClpP is strikingly analogous to that of eukaryotic and archaeal 20S proteasomes, which is composed of 2 heptameric β-subunit rings enclosing the active site sandwiched between 2 heptameric rings of α subunits. Another protease HslV (ClpQ), also known as the ‘bacterial proteasome’, exhibits analogous packing except that the cylinder is composed of two hexameric rings. The similarity in the cylindrical structural arrangement probably underlines the commonality in the mechanism of function of these proteases. Efforts currently underway in several laboratories should further enhance our understanding of the mechanism of function of these degradative machines. These ongoing efforts might have direct clinical consequences as ClpP has now been shown to be a legitimate antibacterial drug target.

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