The 20S proteasome is regulated at multiple levels including association with endogenous activators. Two activators have been described for the yeast 20S proteasome: the 19S regulatory particle and the Blm10 protein. The sequence of Blm10 is 20% identical to the mammalian PA200 protein. Recent studies have shown that the sequences of Blm10 and PA200 each contain multiple HEAT-repeats and that each binds to the ends of mature proteasomes, suggesting a common structural and biochemical function. In order to advance structural studies, we have developed an efficient purification method that produces high yields of stoichiometric Blm10-mature yeast 20S proteasome complexes and we constructed a three-dimensional (3D) model of the Blm10-20S complex from cryo-electron microscopy images. This reconstruction shows that Blm10 binds in a defined orientation to both ends of the 20S particle and contacts all the proteasome α subunits. Blm10 displays the solenoid folding predicted by the presence of multiple HEAT-like repeats and the axial gates on the α rings of the proteasome appear to be open in the complex. We also performed a genetic analysis in an effort to identify the physiological role of Blm10. These experiments, however, did not reveal a robust phenotype upon gene deletion, overexpression, or in a screen for synthetic effects. This leaves the physiological role of Blm10 unresolved, but challenges earlier findings of a role in DNA repair.

The Escherichia coli DegP protein is an essential periplasmic protein for bacterial survival at high temperatures. DegP has the unusual property of working as a chaperone below 28°C, but efficiently degrading unfolded proteins above 28°C. Monomeric DegP contains a protease domain and two PDZ domains. It oligomerizes into a hexameric cage through the staggered association of trimers. The active sites are located in a central cavity that is only accessible laterally, and the 12 PDZ domains act as mobile sidewalls that mediate opening and closing of the gates. As access to the active sites is restricted, DegP is an example of a selfcompartmentalized protease. To determine the essential elements of DegP that maintain the integrity of the hexameric cage, we constructed several deletion mutants of DegP that formed trimers rather than hexamers. We found that residues 39 to 78 within the LA loops, as well as the PDZ2 domains are essential for the integrity of the DegP hexamer. In addition, we asked whether an enclosed cavity or cage of specific dimensions is required for the protease and chaperone activities in DegP. Both activities were maintained in the trimeric DegP mutants without an enclosed cavity and in deletion DegP mutants with significantly reduced dimensions of the cage. We conclude that the functional unit for the protease and chaperone activities of DegP is a trimer and that neither a cavity of specific dimensions nor the presence of an enclosed cavity appears to be essential for the protease and chaperone activities of DegP.Heat and chemical stress produce damaged and unfolded proteins that are highly toxic to cells (27). The periplasm of Escherichia coli is more susceptible to stress factors because it is separated from the environment only by a porous outer membrane (17). DegP (also called HtrA or protease Do) (14, 24) is an essential protein for E. coli (13), as it eliminates unfolded and damaged proteins that would otherwise form aggregates and thus compromise the survival of the cell (3).Chaperones and proteases perform antagonistic functions, but also often work together, as demonstrated by the Clp protein family in which the peptidase ClpP acts together with the chaperone ClpA or ClpX (5, 9, 16). DegP has the unusual property of working as a chaperone below 28°C but efficiently degrading unfolded proteins above 28°C. Thus, DegP is an example of both chaperone and protease functions combined in the same protein (23). Also, unlike other chaperones and proteases whose activities usually require expenditure of ATP, these activities are carried out in the absence of metabolic energy in DegP (10, 23).The mature DegP monomer (48 kDa) contains a trypsin-like protease domain at the N terminus, followed by two PDZ domains (11). PDZ domains are modular interaction domains involved in protein targeting and protein complex assembly that bind preferentially to the C-terminal three to four residues of their target protein. The structure of DegP determined by X-ray crystallography (Protein Data Bank [PDB] identific...

PDZ domains are modular protein interaction domains that are present in metazoans and bacteria. These domains possess unique structural features that allow them to interact with the C-terminal residues of their ligands. The Escherichia coli essential periplasmic protein DegP contains two PDZ domains attached to the C-terminal end of the protease domain. In this study we examined the role of each PDZ domain in the protease and chaperone activities of this protein. Specifically, DegP mutants with either one or both PDZ domains deleted were generated and tested to determine their protease and chaperone activities, as well as their abilities to sequester unfolded substrates. We found that the PDZ domains in DegP have different roles; the PDZ1 domain is essential for protease activity and is responsible for recognizing and sequestering unfolded substrates through C-terminal tags, whereas the PDZ2 domain is mostly involved in maintaining the hexameric cage of DegP. Interestingly, neither of the PDZ domains was required for the chaperone activity of DegP. In addition, we found that the loops connecting the protease domain to PDZ1 and connecting PDZ1 to PDZ2 are also essential for the protease activity of the hexameric DegP protein. New insights into the roles of the PDZ domains in the structure and function of DegP are provided. These results imply that DegP recognizes substrate molecules targeted for degradation and substrate molecules targeted for refolding in different manners and suggest that the substrate recognition mechanisms may play a role in the protease-chaperone switch, dictating whether the substrate is degraded or refolded.PDZ domains represent a common protein interaction motif, and their name was derived from the first three proteins in which such domains were identified, namely PSD-95, Drosophila melanogaster Disc large protein, and zonula occludens protein 1. Bacterial PDZ domains are homologous to the metazoan PDZ domains (23,24,26); however, their topology is different (20), and thus they are designated "PDZ-like" domains.PDZ domains are approximately 90 residues long and have a common structure consisting of six ␤-strands and two ␣-helices, which fold in an overall six-stranded ␤-sandwich. The C-terminal ends of a protein substrate (7) usually bind in a groove of the domain formed between one of the ␣-helices and the adjacent ␤-strand, which thus serves as an extra ␤-strand added to the ␤-sheet (8). In this manner, the C-terminal peptide backbone participates in an extensive hydrogen-bonding pattern with the main chain atoms of the PDZ domain ␤-strand. The terminal carboxylate group is also stabilized by a series of hydrogen bonds with the highly conserved "carboxylate-binding loop" (3). However, the side chain of the C-terminal residue and the side chain of the residue at position Ϫ2 are the structures that are most critical for the specificity of substrate recognition by the PDZ domain, rather than the extensive hydrogen bonds with the main chain of the PDZ domain ␤-strand (29). In this context, ...

The Escherichia coli HtrA protein is a periplasmic protease/chaperone that is upregulated under stress conditions. The protease and chaperone activities of HtrA eliminate or refold damaged and unfolded proteins in the bacterial periplasm that are generated upon stress conditions. In the absence of substrates, HtrA oligomerizes into a hexameric cage, but binding of misfolded proteins transforms the hexamers into bigger 12-mer and 24-mer cages that encapsulate the substrates for degradation or refolding. HtrA also undergoes partial degradation as a consequence of self-cleavage of the mature protein, producing short-HtrA protein (s-HtrA). The aim of this study was to examine the physiological role of this self-cleavage process. We found that the only requirement for self-cleavage of HtrA into s-HtrA in vitro was the hydrolysis of protein substrates. In fact, peptides resulting from the hydrolysis of the protein substrates were sufficient to induce autocleavage. However, the continuous presence of full-length substrate delayed the process. In addition, we observed that the hexameric cage structure is required for autocleavage and that s-HtrA accumulates only late in the degradation reaction. These results suggest that self-cleavage occurs when HtrA reassembles back into the resting hexameric structure and peptides resulting from substrate hydrolysis are allosterically stimulating the HtrA proteolytic activity. Our data support a model in which the physiological role of the self-cleavage process is to eliminate the excess of HtrA once the stress conditions cease.

Structural biology has been extremely successful in providing functional insights for a large number of proteins and macromolecular assemblies, but in some cases, the structure has contributed a three-dimensional (3D) framework to interpret years of accumulated biochemical and genetic knowledge. In these particular systems, structural information has allowed us to learn things that would have been difficult to learn with other techniques.The periplasmic Escherichia coli DegP protease/chaperone exemplifies this scenario very well. This protein was initially identified in the 1980s (23,24,37,38), and over more than two decades, several groups characterized its activities (2,12,16,27). However, a comprehensive 3D functional model did not become apparent until the first DegP X-ray structure was revealed in 2002 (20). Following the discovery of this remarkable structure, a number of groups concentrated on testing the essentials of the functional model proposed from the crystal structure. Interestingly, this functional model was rewritten recently, after the structures of DegP in two additional oligomeric forms were resolved (13,22). Current research efforts are now concentrated on probing the new functional model and also on answering new, interesting questions posed by these structures. Therefore, DegP provides an excellent example for the structure-driven study of protein function. This minireview aims to summarize how the functional model for DegP protein has evolved as the structures of the different oligomeric forms of the protein have been elucidated.E. coli DegP (also called HtrA or protease Do) is an important periplasmic protein with the unusual property of functioning both as a protease and as a chaperone (36). Unlike the cytoplasmic compartment, the periplasm lacks ATP and does not support the function of large protein machines powered by this molecule. However, in this cellular compartment, DegP can still degrade and refold misfolded proteins in an ATPindependent manner (2). Although DegP is not an essential protein, its activity is required for bacterial survival at high temperatures (34) and under harsh environmental conditions. Consequently, its expression is upregulated by both the Cpx and E protein quality control pathways under conditions of protein-folding stress (3, 29).DegP homologs have been isolated from a variety of species, including gram-negative and -positive bacteria, plants, and mammals. All these proteins constitute the HtrA family of proteases (2). In bacteria, members of this family are key players mainly in protein quality control in the periplasmic space. In eukaryotic cells, these proteins are involved in functions as diverse as the regulation of apoptosis (1, 4, 9) and the delay of the aggregation process of intracellular amyloid peptides (19). HtrA proteins usually contain a protease domain and at least one C-terminal PDZ domain. In some cases, members of this family of proteins also include additional domains, such as transmembrane regions, located usually at the N terminus. Spec...