Tricorn-like proteases in bacteria

The tricorn protease is an archaeal protease that forms massive proteasome-like capsids with a hollow chamber. beta-Propeller and PDZ domains are thought to play a role in substrate selection. By analysis of predicted proteins from novel bacterial genome sequences, we have identified four new bacterial tricorn-like proteases, complete with similar beta-propeller, PDZ and catalytic domains. We propose various hypotheses as to the function of these domains that can now be tested in the laboratory.     

Crystal structures of the tricorn interacting factor F3 from Thermoplasma acidophilum, a zinc aminopeptidase in three different conformations

The tricorn interacting factor F3 is an 89 kDa zinc aminopeptidase from the archaeon Thermoplasma acidophilum. Together with the tricorn interacting factors F1 and F2, F3 degrades the tricorn protease products and thus completes the proteasomal degradation pathway by generating free amino acids. Here, we present the crystal structures of F3 in three different conformations at 2.3 A resolution. The zinc aminopeptidase is composed of four domains: an N-terminal saddle-like beta-structure domain; a thermolysin-like catalytic domain; a small barrel-like beta-structure domain; and an alpha-helical C-terminal domain, the latter forming a deep cavity at the active site. Three crystal forms provide snapshots of the molecular dynamics of F3 where the C-terminal domain can adapt to form an open, an intermediate and a nearly closed cavity, respectively. With the conserved Zn(2+)-binding motifs HEXXH and NEXFA as well as the N-terminal substrate-anchoring glutamate residues, F3 together with the leukotriene A4 hydrolase, represents a novel gluzincin subfamily of aminoproteases. We discuss the functional implications of these structures with respect to the underlying catalytic mechanism, substrate recognition and processing, and possible component interactions.     

Evidence for PDZ domains in bacteria, yeast, and plants.

Several dozen signaling proteins are now known to contain 80-100 residue repeats, called PDZ (or DHR or GLGF) domains, several of which interact with the C-terminal tetrapeptide motifs X-Ser/Thr-X-Val-COO- of ion channels and/or receptors. PDZ domains have previously been noted only in mammals, flies, and worms, suggesting that the primordial PDZ domain arose relatively late in eukaryotic evolution. Here, techniques of sequence analysis-including local alignment, profile, and motif database searches-indicate that PDZ domain homologues are present in yeast, plants, and bacteria. It is suggested that two PDZ domains occur in bacterial high-temperature requirement A (htrA) and one in tail-specific protease (tsp) homologues, and that a yeast htrA homologue contains four PDZ domains. Sequence comparisons suggest that the spread of PDZ domains in these diverse organisms may have occurred via horizontal gene transfer. The known affinity of Escherichia coli tsp for C-terminal polypeptides is proposed to be mediated by its PDZ-like domain, in a similar manner to the binding of C-terminal polypeptides by animal PDZ domains.     

Cryo-EM structure of hexameric yeast Lon protease (PIM1) highlights the importance of conserved structural elements

Lon protease is a conserved ATP-dependent serine protease composed of an AAA+ domain that mechanically unfolds substrates and a serine protease domain that degrades these unfolded substrates. In yeast, dysregulation of Lon protease (PIM1) attenuates lifespan and leads to gross mitochondrial morphological perturbations. Although structures of the bacterial and human Lon protease reveal a hexameric assembly, yeast PIM1 was speculated to form a heptameric assembly and is uniquely characterized by a ∼50-residue insertion between the ATPase and protease domains. To further understand the yeast-specific properties of PIM1, we determined a high-resolution cryo-electron microscopy structure of PIM1 in a substrate-translocating state. Here, we reveal that PIM1 forms a hexamer, conserved with that of bacterial and human Lon proteases, wherein the ATPase domains form a canonical closed spiral that enables pore loop residues to translocate substrates to the protease chamber. In the substrate-translocating state, PIM1 protease domains form a planar protease chamber in an active conformation and are uniquely characterized by a ∼15-residue C-terminal extension. These additional C-terminal residues form an α-helix located along the base of the protease domain. Finally, we did not observe density for the yeast-specific insertion between the ATPase and protease domains, likely due to high conformational flexibility. Biochemical studies to investigate the insertion using constructs that truncated or replaced the insertion with a glycine-serine linker suggest that the yeast-specific insertion is dispensable for PIM1’s enzymatic function. Altogether, our structural and biochemical studies highlight unique components of PIM1 machinery and demonstrate evolutionary conservation of Lon protease function.     

Structural and functional analysis of the ligand specificity of the HtrA2/Omi PDZ domain

The mitochondrial serine protease HtrA2/Omi helps to maintain mitochondrial function by handling misfolded proteins in the intermembrane space. In addition, HtrA2/Omi has been implicated as a proapoptotic factor upon release into the cytoplasm during the cell death cascade. The protein contains a C-terminal PDZ domain that packs against the protease active site and inhibits proteolytic activity. Engagement of the PDZ domain by peptide ligands has been shown to activate the protease and also has been proposed to mediate substrate recognition. We report a detailed structural and functional analysis of the human HtrA2/Omi PDZ domain using peptide libraries and affinity assays to define specificity, X-ray crystallography to view molecular details of PDZ-ligand interactions, and alanine-scanning mutagenesis to probe the peptide-binding groove. We show that the HtrA2/Omi PDZ domain recognizes both C-terminal and internal stretches of extended, hydrophobic polypeptides. High-affinity ligand recognition requires contacts with up to five hydrophobic side chains by distinct sites on the PDZ domain. However, no particular residue type is absolutely required at any position, and thus, the HtrA2/Omi PDZ domain appears to be a promiscuous module adapted to recognize unstructured, hydrophobic polypeptides. This type of specificity is consistent with the biological role of HtrA2/Omi in mitochondria, which requires the recognition of diverse, exposed stretches of hydrophobic sequences in misfolded proteins. The findings are less consistent with, but do not exclude, a role for the PDZ domain in targeting the protease to specific substrates during apoptosis.     

Slicing a protease: structural features of the ATP-dependent Lon proteases gleaned from investigations of isolated domains

ATP-dependent Lon proteases are multi-domain enzymes found in all living organisms. All Lon proteases contain an ATPase domain belonging to the AAA(+) superfamily of molecular machines and a proteolytic domain with a serine-lysine catalytic dyad. Lon proteases can be divided into two subfamilies, LonA and LonB, exemplified by the Escherichia coli and Archaeoglobus fulgidus paralogs, respectively. The LonA subfamily is defined by the presence of a large N-terminal domain, whereas the LonB subfamily has no such domain, but has a membrane-spanning domain that anchors the protein to the cytoplasmic side of the membrane. The two subfamilies also differ in their consensus sequences. Recent crystal structures for several individual domains and sub-fragments of Lon proteases have begun to illuminate similarities and differences in structure-function relationships between the two subfamilies. Differences in orientation of the active site residues in several isolated Lon protease domains point to possible roles for the AAA(+) domains and/or substrates in positioning the catalytic residues within the active site. Structures of the proteolytic domains have also indicated a possible hexameric arrangement of subunits in the native state of bacterial Lon proteases. The structure of a large segment of the N-terminal domain has revealed a folding motif present in other protein families of unknown function and should lead to new insights regarding ways in which Lon interacts with substrates or other cellular factors. These first glimpses of the structure of Lon are heralding an exciting new era of research on this ancient family of proteases.     

Structures of the tricorn-interacting aminopeptidase F1 with different ligands explain its catalytic mechanism

F1 is a 33.5 kDa serine peptidase of the alpha/beta-hydrolase family from the archaeon Thermoplasma acidophilum. Subsequent to proteasomal protein degradation, tricorn generates small peptides, which are cleaved by F1 to yield single amino acids. We have solved the crystal structure of F1 with multiwavelength anomalous dispersion (MAD) phasing at 1.8 A resolution. In addition to the conserved catalytic domain, the structure reveals a chiefly alpha-helical domain capping the catalytic triad. Thus, the active site is accessible only through a narrow opening from the protein surface. Two structures with molecules bound to the active serine, including the inhibitor phenylalanyl chloromethylketone, elucidate the N-terminal recognition of substrates and the catalytic activation switch mechanism of F1. The cap domain mainly confers the specificity for hydrophobic side chains by a novel cavity system, which, analogously to the tricorn protease, guides substrates to the buried active site and products away from it. Finally, the structure of F1 suggests a possible functional complex with tricorn that allows efficient processive degradation to free amino acids for cellular recycling.     

Role of the PDZ domains in Escherichia coli DegP protein

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.     

A new principle of oligomerization of plant DEG7 protease based on interactions of degenerated protease domains

Deg/HtrA proteases are a large group of ATP-independent serine endoproteases found in almost every organism. Their usual domain arrangement comprises a trypsin-type protease domain and one or more PDZ domains. All Deg/HtrA proteases form homo-oligomers with trimers as the basic unit, where the active protease domain mediates the interaction between individual monomers. Among the members of the Deg/HtrA protease family, the plant protease DEG7 is unique since it contains two protease domains (one active and one degenerated) and four PDZ domains. In the present study, we investigated the oligomerization behaviour of this unusual protease using yeast two-hybrid analysis in vivo and with recombinant protein in vitro. We show that DEG7 forms trimeric complexes, but in contrast with other known Deg/HtrA proteases, it shows a new principle of oligomerization, where trimerization is based on the interactions between degenerated protease domains. We propose that, during evolution, a duplicated active protease domain degenerated and specialized in protein-protein interaction and complex formation.     

Structural and functional analysis of the PDZ domains of human HtrA1 and HtrA3

High-temperature requirement A (HtrA) and its homologs contain a serine protease domain followed by one or two PDZ domains. Bacterial HtrA proteins and the mitochondrial protein HtrA2/Omi maintain cell function by acting as both molecular chaperones and proteases to manage misfolded proteins. The biological roles of the mammalian family members HtrA1 and HtrA3 are less clear. We report a detailed structural and functional analysis of the PDZ domains of human HtrA1 and HtrA3 using peptide libraries and affinity assays to define specificity, structural studies to view the molecular details of ligand recognition, and alanine scanning mutagenesis to investigate the energetic contributions of individual residues to ligand binding. In common with HtrA2/Omi, we show that the PDZ domains of HtrA1 and HtrA3 recognize hydrophobic polypeptides, and while C-terminal sequences are preferred, internal sequences are also recognized. However, the details of the interactions differ, as different domains rely on interactions with different residues within the ligand to achieve high affinity binding. The results suggest that mammalian HtrA PDZ domains interact with a broad range of hydrophobic binding partners. This promiscuous specificity resembles that of bacterial HtrA family members and suggests a similar function for recognizing misfolded polypeptides with exposed hydrophobic sequences. Our results support a common activation mechanism for the HtrA family, whereby hydrophobic peptides bind to the PDZ domain and induce conformational changes that activate the protease. Such a mechanism is well suited to proteases evolved for the recognition and degradation of misfolded proteins.     

Structural and Functional Analysis of Human HtrA3 Protease and Its Subdomains

Human HtrA3 protease, which induces mitochondria-mediated apoptosis, can be a tumor suppressor and a potential therapeutic target in the treatment of cancer. However, there is little information about its structure and biochemical properties. HtrA3 is composed of an N-terminal domain not required for proteolytic activity, a central serine protease domain and a C-terminal PDZ domain. HtrA3S, its short natural isoform, lacks the PDZ domain which is substituted by a stretch of 7 C-terminal amino acid residues, unique for this isoform. This paper presents the crystal structure of the HtrA3 protease domain together with the PDZ domain (ΔN-HtrA3), showing that the protein forms a trimer whose protease domains are similar to those of human HtrA1 and HtrA2. The ΔN-HtrA3 PDZ domains are placed in a position intermediate between that in the flat saucer-like HtrA1 SAXS structure and the compact pyramidal HtrA2 X-ray structure. The PDZ domain interacts closely with the LB loop of the protease domain in a way not found in other human HtrAs. ΔN-HtrA3 with the PDZ removed (ΔN-HtrA3-ΔPDZ) and an N-terminally truncated HtrA3S (ΔN-HtrA3S) were fully active at a wide range of temperatures and their substrate affinity was not impaired. This indicates that the PDZ domain is dispensable for HtrA3 activity. As determined by size exclusion chromatography, ΔN-HtrA3 formed stable trimers while both ΔN-HtrA3-ΔPDZ and ΔN-HtrA3S were monomeric. This suggests that the presence of the PDZ domain, unlike in HtrA1 and HtrA2, influences HtrA3 trimer formation. The unique C-terminal sequence of ΔN-HtrA3S appeared to have little effect on activity and oligomerization. Additionally, we examined the cleavage specificity of ΔN-HtrA3. Results reported in this paper provide new insights into the structure and function of ΔN-HtrA3, which seems to have a unique combination of features among human HtrA proteases.     

X-ray snapshots of peptide processing in mutants of tricorn-interacting factor F1 from Thermoplasma acidophilum

The tricorn-interacting factor F1 of the archaeon Thermoplasma acidophilum cleaves small hydrophobic peptide products of the proteasome and tricorn protease. F1 mutants of the active site residues that are involved in substrate recognition and catalysis displayed distinct activity patterns toward fluorogenic test substrates. Crystal structures of the mutant proteins complexed with peptides Phe-Leu, Pro-Pro, or Pro-Leu-Gly-Gly showed interaction of glutamates 213 and 245 with the N termini of the peptides and defined the S1 and S1' sites and the role of the catalytic residues. Evidence was found for processive peptide cleavage in the N-to-C direction, whereby the P1' product is translocated into the S1 site. A functional interaction of F1 with the tricorn protease was observed with the inactive F1 mutant G37A. Moreover, small angle x-ray scattering measurements for tricorn and inhibited F1 have been interpreted as formation of transient and substrate-induced complexes.     

Site-2 protease regulated intramembrane proteolysis: sequence homologs suggest an ancient signaling cascade

Site-2 proteases (S2Ps) form a large family of membrane-embedded metalloproteases that participate in cellular signaling pathways through sequential cleavage of membrane-tethered substrates. Using sequence similarity searches, we extend the S2P family to include remote homologs that help define a conserved structural core consisting of three predicted transmembrane helices with traditional metalloprotease functional motifs and a previously unrecognized motif (GxxxN/S/G). S2P relatives were identified in genomes from Bacteria, Archaea, and Eukaryota including protists, plants, fungi, and animals. The diverse S2P homologs divide into several groups that differ in various inserted domains and transmembrane helices. Mammalian S2P proteases belong to the major ubiquitous group and contain a PDZ domain. Sequence and structural analysis of the PDZ domain support its mediating the sequential cleavage of membrane-tethered substrates. Finally, conserved genomic neighborhoods of S2P homologs allow functional predictions for PDZ-containing transmembrane proteases in extra-cytoplasmic stress response and lipid metabolism.     

Tricorn protease in bacteria: characterization of the enzyme from Streptomyces coelicolor

Tricorn protease is believed to act downstream of the proteasome, or of other ATP-dependent proteases, cleaving the oligopeptides (mostly 6 to 12 residues) released by them into small peptides (2 to 4 residues), before an array of aminopeptidases finally converts them into free amino acids. Hitherto, the occurrence of Tricorn protease seemed to be limited to some archaea, but genes encoding Tricorn homologs have now been found in several bacterial genomes. Among them is Streptomyces coelicolor A3(2), which has, in fact, two Tricorn-like genes, ScC77.16c and ScE87.19. The proteins encoded by them (TRI-ScC77 and TRI-ScE87) are very similar in their PDZ and TSP domains, but rather divergent in their beta-propeller domains. We have expressed one of them, TRI-ScC77, in E. coil and characterized the recombinant protein structurally and functionally. TRI-ScC77 forms a homohexameric complex of approximately 700 kDa, both in E. coil and in S. coelicolor, with enzymatic properties very similar to the complex from the archaeon Thermoplasma acidophilum. The fact that Tricorn-like proteins exist not only in thermoacidophiles, but also in bacteria inhabiting radically different environments, rules out the possibility that Tricorn protease is an adaptive element that helps to meet the challenges of an extreme habitat.     

The structure and function of human dipeptidyl peptidase IV,possessing a unique eight-bladed beta-propeller fold

Dipeptidyl peptidase IV (DPPIV) is a serine protease, a member of the prolyl oligopeptidase (POP) family, and has been implicated in several diseases. Therefore, the development of DPPIV selective inhibitors, which are able to control the biological function of DPPIV, is important. We determined the crystal structure of human DPPIV at 2.6A resolution. The molecule consists of a unique eight-bladed beta-propeller domain in the N-terminal region and a serine protease domain in the C-terminal region. Also, the large "cave" structure, which is thought to control the access of the substrate, is found on the side of the beta-propeller fold. Comparison of the overall amino acid sequence between human DPPIV and POP shows low homology (12.9%). In this paper, we report the structure of human DPPIV, especially focusing on a unique eight-bladed beta-propeller domain. We also discuss the way for the access of the substrate to this domain.     

The Thermoplasma acidophilum Lon protease has a Ser-Lys dyad active site.

A gene with significant similarity to bacterial Lon proteases was identified during the sequencing of the genome of the thermoacidophilic archaeon Thermoplasma acidophilum. Protein sequence comparison revealed that Thermoplasma Lon protease (TaLon) is more similar to the LonB proteases restricted to Gram-positive bacteria than to the widely distributed bacterial LonA. However, the active site residues of the protease and ATPase domain are highly conserved in all Lon proteases. Using site-directed mutagenesis we show here that TaLon and EcLon, and probably all other Lon proteases, contain a Ser-Lys dyad active site. The TaLon active site mutants were fully assembled and, similar to TaLon wild-type, displayed an apparent molar mass of 430 kDa upon gelfiltration. This would be consistent with a hexameric complex and indeed electron micrographs of TaLon revealed ring-shaped particles, although of unknown symmetry. Comparison of the ATPase activity of Lon wild-type from Thermoplasma or Escherichia coli with respective protease active site mutants revealed differences in Km and V values. This suggests that in the course of protein degradation by wild-type Lon the protease domain might influence the activity of the ATPase domain.     

GlyGly-CTERM and rhombosortase: a C-terminal protein processing signal in a many-to-one pairing with a rhomboid family intramembrane serine protease

The rhomboid family of serine proteases occurs in all domains of life. Its members contain at least six hydrophobic membrane-spanning helices, with an active site serine located deep within the hydrophobic interior of the plasma membrane. The model member GlpG from Escherichia coli is heavily studied through engineered mutant forms, varied model substrates, and multiple X-ray crystal studies, yet its relationship to endogenous substrates is not well understood. Here we describe an apparent membrane anchoring C-terminal homology domain that appears in numerous genera including Shewanella, Vibrio, Acinetobacter, and Ralstonia, but excluding Escherichia and Haemophilus. Individual genomes encode up to thirteen members, usually homologous to each other only in this C-terminal region. The domain's tripartite architecture consists of motif, transmembrane helix, and cluster of basic residues at the protein C-terminus, as also seen with the LPXTG recognition sequence for sortase A and the PEP-CTERM recognition sequence for exosortase. Partial Phylogenetic Profiling identifies a distinctive rhomboid-like protease subfamily almost perfectly co-distributed with this recognition sequence. This protease subfamily and its putative target domain are hereby renamed rhombosortase and GlyGly-CTERM, respectively. The protease and target are encoded by consecutive genes in most genomes with just a single target, but far apart otherwise. The signature motif of the Rhombo-CTERM domain, often SGGS, only partially resembles known cleavage sites of rhomboid protease family model substrates. Some protein families that have several members with C-terminal GlyGly-CTERM domains also have additional members with LPXTG or PEP-CTERM domains instead, suggesting there may be common themes to the post-translational processing of these proteins by three different membrane protein superfamilies.     

The serine protease HhoA from Synechocystis sp. strain PCC 6803: substrate specificity and formation of a hexameric complex are regulated by the PDZ domain

Enzymes of the ATP-independent Deg serine endopeptidase family are very flexible with regard to their substrate specificity. Some family members cleave only one substrate, while others act as general proteases on unfolded substrates. The proteolytic activity of Deg proteases is regulated by PDZ protein interaction domains. Here we characterized the HhoA protease from Synechocystis sp. strain PCC 6803 in vitro using several recombinant protein constructs. The proteolytic activity of HhoA was found to increase with temperature and basic pH and was stimulated by the addition of Mg(2+) or Ca(2+). We found that the single PDZ domain of HhoA played a critical role in regulating protease activity and in the assembly of a hexameric complex. Deletion of the PDZ domain strongly reduced proteolysis of a sterically challenging resorufin-labeled casein substrate, but unlabeled beta-casein was still degraded. Reconstitution of the purified HhoA with total membrane proteins isolated from Synechocystis sp. wild-type strain PCC 6803 and a DeltahhoA mutant resulted in specific degradation of selected proteins at elevated temperatures. We concluded that a single PDZ domain of HhoA plays a critical role in defining the protease activity and oligomerization state, combining the functions that are attributed to two PDZ domains in the homologous DegP protease from Escherichia coli. Based on this first enzymatic study of a Deg protease from cyanobacteria, we propose a general role for HhoA in the quality control of extracytoplasmic proteins, including membrane proteins, in Synechocystis sp. strain PCC 6803.