| Abstract: | ![]()
The Clp protease is conserved among eubacteria and most eukaryotes, and uses ATP to drive protein substrate unfolding and translocation into a chamber of sequestered proteolytic active sites. The main constitutive Clp protease in photosynthetic organisms has evolved into a functionally essential and structurally intricate enzyme. The model Clp protease from the cyanobacterium Synechococcus consists of the HSP100 molecular chaperone ClpC and a mixed proteolytic core comprised of two distinct subunits, ClpP3 and ClpR. We have purified the ClpP3/R complex, the first for a Clp proteolytic core comprised of heterologous subunits. The ClpP3/R complex has unique functional and structural features, consisting of twin heptameric rings each with an identical ClpP33ClpR4 configuration. As predicted by its lack of an obvious catalytic triad, the ClpR subunit is shown to be proteolytically inactive. Interestingly, extensive modification to ClpR to restore proteolytic activity to this subunit showed that its presence in the core complex is not rate-limiting for the overall proteolytic activity of the ClpCP3/R protease. Altogether, the ClpP3/R complex shows remarkable similarities to the 20 S core of the proteasome, revealing a far greater degree of convergent evolution than previously thought between the development of the Clp protease in photosynthetic organisms and that of the eukaryotic 26 S proteasome.Proteases perform numerous tasks vital for cellular homeostasis in all organisms. Much of the selective proteolysis within living cells is performed by multisubunit chaperone-protease complexes. These proteases all share a common two-component architecture and mode of action, with one of the best known examples being the proteasome in archaebacteria, certain eubacteria, and eukaryotes (1).The 20 S proteasome is a highly conserved cylindrical structure composed of two distinct types of subunits, α and β. These are organized in four stacked heptameric rings, with two central β-rings sandwiched between two outer α-rings. Although the α- and β-protein sequences are similar, it is only the latter that is proteolytic active, with a single Thr active site at the N terminus. The barrel-shaped complex is traversed by a central channel that widens up into three cavities. The catalytic sites are positioned in the central chamber formed by the β-rings, adjacent to which are two antechambers conjointly built up by β- and α-subunits. In general, substrate entry into the core complex is essentially blocked by the α-rings, and thus relies on the associating regulatory partner, PAN and 19 S complexes in archaea and eukaryotes, respectively (1). Typically, the archaeal core structure is assembled from only one type of α- and β-subunit, so that the central proteolytic chamber contains 14 catalytic active sites (2). In contrast, each ring of the eukaryotic 20 S complex has seven distinct α- and β-subunits. Moreover, only three of the seven β-subunits in each ring are proteolytically active (3). Having a strictly conserved architecture, the main difference between the 20 S proteasomes is one of complexity. In mammalian cells, the three constitutive active subunits can even be replaced with related subunits upon induction by γ-interferon to generate antigenic peptides presented by the class 1 major histocompatibility complex (4).Two chambered proteases architecturally similar to the proteasome also exist in eubacteria, HslV and ClpP. HslV is commonly thought to be the prokaryotic counterpart to the 20 S proteasome mainly because both are Thr proteases. A single type of HslV protein, however, forms a proteolytic chamber consisting of twin hexameric rather than heptameric rings (5). Also displaying structural similarities to the proteasome is the unrelated ClpP protease. The model Clp protease from Escherichia coli consists of a proteolytic ClpP core flanked on one or both sides by the ATP-dependent chaperones ClpA or ClpX (6). The ClpP proteolytic chamber is comprised of two opposing homo-heptameric rings with the catalytic sites harbored within (7). ClpP alone displays only limited peptidase activity toward short unstructured peptides (8). Larger native protein substrates need to be recognized by ClpA or ClpX and then translocated in an unfolded state into the ClpP proteolytic chamber (9, 10). Inside, the unfolded substrate is bound in an extended manner to the catalytic triads (Ser-97, His-122, and Asp-171) and degraded into small peptide fragments that can readily diffuse out (11). Several adaptor proteins broaden the array of substrates degraded by a Clp protease by binding to the associated HSP100 partner and modifying its protein substrate specificity (12, 13). One example is the adaptor ClpS that interacts with ClpA (EcClpA) and targets N-end rule substrates for degradation by the ClpAP protease (14).Like the proteasome, the Clp protease is found in a wide variety of organisms. Besides in all eubacteria, the Clp protease also exist in mammalian and plant mitochondria, as well as in various plastids of algae and plants. It also occurs in the unusual plastid in Apicomplexan protozoan (15), a family of parasites responsible for many important medical and veterinary diseases such as malaria. Of all these organisms, photobionts have by far the most diverse array of Clp proteins. This was first apparent in cyanobacteria, with the model species Synechococcus elongatus having 10 distinct Clp proteins, four HSP100 chaperones (ClpB1–2, ClpC, and ClpX), three ClpP proteins (ClpP1–3), a ClpP-like protein termed ClpR, and two adaptor proteins (ClpS1–2) (16). Of particular interest is the ClpR variant, which has protein sequence similarity to ClpP but appears to lack the catalytic triad of Ser-type proteases (17). This diversity of Clp proteins is even more extreme in photosynthetic eukaryotes, with at least 23 different Clp proteins in the higher plant Arabidopsis thaliana, most of which are plastid-localized (18).We have recently shown that two distinct Clp proteases exist in Synechococcus, both of which contain mixed proteolytic cores. The first consists of ClpP1 and ClpP2 subunits, and associates with ClpX, whereas the other has a proteolytic core consisting of ClpP3 and ClpR that binds to ClpC, as do the two ClpS adaptors (19). Of these proteases, it is the more constitutively abundant ClpCP3/R that is essential for cell viability and growth (20, 21). It is also the ClpP3/R complex that is homologous to the single type in eukaryotic plastids, all of which also have ClpC as the chaperone partner (16). In algae and plants, however, the complexity of the plastidic Clp proteolytic core has evolved dramatically. In Arabidopsis, the core complex consists of five ClpP and four ClpR paralogs, along with two unrelated Clp proteins unique to higher plants (22). Like ClpP3/R, the plastid Clp protease in Arabidopsis is essential for normal growth and development, and appears to function primarily as a housekeeping protease (23, 24).One of the most striking developments in the Clp protease in photosynthetic organisms and Apicomplexan parasites is the inclusion of ClpR within the central proteolytic core. Although this type of Clp protease has evolved into a vital enzyme, little is known about its activity or the exact role of ClpR within the core complex. To address these points we have purified the intact Synechococcus ClpP3/R proteolytic core by co-expression in E. coli. The recombinant ClpP3/R forms a double heptameric ring complex, with each ring having a specific ClpP3/R stoichiometry and arrangement. Together with ClpC, the ClpP3/R complex degrades several polypeptide substrates, but at a rate considerably slower than that by the E. coli ClpAP protease. Interestingly, although ClpR is shown to be proteolytically inactive, its inclusion in the core complex is not rate-limiting to the overall activity of the ClpCP3/R protease. In general, the results reveal remarkable similarities between the evolutionary development of the Clp protease in photosynthetic organisms and the eukaryotic proteasome relative to their simpler prokaryotic counterparts. |
