Concerted structural changes in the peptidase and the propeller domains of prolyl oligopeptidase are required for substrate binding

Prolyl oligopeptidase contains a peptidase domain and its catalytic triad is covered by the central tunnel of a seven-bladed beta-propeller. This domain makes the enzyme an oligopeptidase by excluding large structured peptides from the active site. The apparently rigid crystal structure does not explain how the substrate can approach the catalytic groups. Two possibilities of substrate access were investigated: either blades 1 and 7 of the propeller domain move apart, or the peptidase and/or propeller domains move to create an entry site at the domain interface. Engineering disulfide bridges to the expected oscillating structures prevented such movements, which destroyed the catalytic activity and precluded substrate binding. This indicated that concerted movements of the propeller and the peptidase domains are essential for the enzyme action.     

Rigidity and flexibility of dipeptidyl peptidase IV: crystal structures of and docking experiments with DPIV

Dipeptidyl peptidase IV (DPIV) is an alpha,beta-hydrolase-like serine exopeptidase, which removes dipeptides, preferentially with a C-terminal l-Pro residue, from the N terminus of longer peptide substrates. Previously, we determined the tetrameric 1.8A crystal structure of native porcine DPIV. Each monomer is composed of a beta-propeller and a catalytic domain, which together embrace an internal cavity housing the active centre. This cavity is connected to the bulk solvent by a "propeller opening" and a "side opening". Here, we analyse DPIV complexes with a t-butyl-Gly-Pro-Ile tripeptide, Pro-boroPro, a piperazine purine compound, and aminoethyl phenyl sulfonylfluoride. The latter two compounds bind to the active-site groove in a compact and a quite bulky manner, respectively, causing considerable shifts of the catalytic Ser630 side-chain and of the Tyr547 phenolic group, which forms the oxyanion hole. The tripeptide, mimicking a peptide substrate, is clamped to the active site through tight interactions via its N-terminal alpha-ammonium group, the P2 carbonyl group, the P1-l-Pro side-chain, the C-terminal carboxylate group, and the stable orthoacid ester amide formed between the scissile peptide carbonyl group and Ser630 O(gamma). This stable trapping of the tripeptide could be due to stabilization of the protonated His740 imidazolium cation by the adjacent negatively charged C-terminal carboxylate group, preventing proton transfer to the leaving group nitrogen atom. Docking experiments with the compact rigid 58 residue protein aprotinin, which had been shown to be processed by DPIV, indicate that the Arg1-Pro2 N terminus can access the DPIV active site only upon widening of its side openings, probably by separation of the first and the last propeller blades, and/or of the catalytic and the propeller domain.     

Catalysis of serine oligopeptidases is controlled by a gating filter mechanism

Proteases have a variety of strategies for selecting substrates in order to prevent uncontrolled protein degradation. A recent crystal structure determination of prolyl oligopeptidase has suggested a way for substrate selection involving an unclosed seven-bladed β-propeller domain. We have engineered a disulfide bond between the first and seventh blades of the propeller, which resulted in the loss of enzymatic activity. These results provided direct evidence for a novel strategy of regulation in which oscillating propeller blades act as a gating filter during catalysis, letting small peptide substrates into the active site while excluding large proteins to prevent accidental proteolysis.     

Flexibility of prolyl oligopeptidase: molecular dynamics and molecular framework analysis of the potential substrate pathways

The flexibility of prolyl oligopeptidase has been investigated using molecular dynamics (MD) and molecular framework approaches to delineate the route of the substrate to the active site. The selectivity of the enzyme is mediated by a seven-bladed beta-propeller that in the crystal structure does not indicate the possible passage for the substrate to the catalytic center. Its open topology however, could allow the blades to move apart and let the substrate into the large central cavity. Flexibility analysis of prolyl oligopeptidase structure using the FIRST (Floppy Inclusion and Rigid Substructure Topology) approach and the atomic fluctuations derived from MD simulations demonstrated the rigidity of the propeller domain, which does not permit the substrate to approach the active site through this domain. Instead, a smaller tunnel at the inter-domain region comprising the highly flexible N-terminal segment of the peptidase domain and a facing hydrophilic loop from the propeller (residues 192-205) was identified by cross-correlation analysis and essential dynamics as the only potential pathway for the substrate. The functional importance of the flexible loop has been also verified by kinetic analysis of the enzyme with a split loop. Catalytic effect of engineered disulfide bridges was rationalized by characterizing the concerted motions of the two domains.     

Crystal structure of human dipeptidyl peptidase IV in complex with a decapeptide reveals details on substrate specificity and tetrahedral intermediate formation

Dipeptidyl peptidase IV (DPPIV) is a member of the prolyl oligopeptidase family of serine proteases. DPPIV removes dipeptides from the N terminus of substrates, including many chemokines, neuropeptides, and peptide hormones. Specific inhibition of DPPIV is being investigated in human trials for the treatment of type II diabetes. To understand better the molecular determinants that underlie enzyme catalysis and substrate specificity, we report the crystal structures of DPPIV in the free form and in complex with the first 10 residues of the physiological substrate, Neuropeptide Y (residues 1-10; tNPY). The crystal structure of the free form of the enzyme reveals two potential channels through which substrates could access the active site-a so-called propeller opening, and side opening. The crystal structure of the DPPIV/tNPY complex suggests that bioactive peptides utilize the side opening unique to DPPIV to access the active site. Other structural features in the active site such as the presence of a Glu motif, a well-defined hydrophobic S1 subsite, and minimal long-range interactions explain the substrate recognition and binding properties of DPPIV. Moreover, in the DPPIV/tNPY complex structure, the peptide is not cleaved but trapped in a tetrahedral intermediate that occurs during catalysis. Conformational changes of S630 and H740 between DPPIV in its free form and in complex with tNPY were observed and contribute to the stabilization of the tetrahedral intermediate. Our results facilitate the design of potent, selective small molecule inhibitors of DPPIV that may yield compounds for the development of novel drugs to treat type II diabetes.     

Truncated prolyl oligopeptidase from Pyrococcus furiosus

The peptidase domain of prolyl oligopeptidase is covered by a propeller domain, which excludes large peptides and proteins from the catalytic triad. Previous studies indicated that some amino acids of the N-terminal region constitute a part of the substrate entrance to the active site. To investigate the catalytic role of the N-terminus, we removed the residues 1-32 from the enzyme and examined the kinetic, thermodynamic, and structural consequences of the deletion, using the thermophile Pyrococcus furiosus prolyl oligopeptidase. An about threefold decrease in the catalytic activity along with a 20 degrees C reduction in the temperature optimum was observed. The pH-rate profile, the rate-limiting step, and the activation parameters did not change significantly. However, a substantial decrease was observed in the stability of the protein as demonstrated by circular dichroism and differential scanning calorimetry measurements, and by denaturation with guanidinium chloride. It was concluded that the N-terminal segment did not facilitate the substrate binding, independent of the size of the substrate, but contributed principally to the protein stability required for the formation of the proper active site.     

Evolution of the beta-propeller fold

beta-Propellers are toroidal folds, in which repeated, four-stranded beta-meanders are arranged in a circular and slightly tilted fashion, like the blades of a propeller. They are found in all domains of life, with a strong preponderance among eukaryotes. Propellers show considerable sequence diversity and are classified into six separate structural groups by the SCOP and CATH databases. Despite this diversity, they often show similarities across groups, not only in structure but also in sequence, raising the possibility of a common origin. In agreement with this hypothesis, most propellers group together in a cluster map of all-beta folds generated by sequence similarity, because of numerous pairwise matches, many of which are individually nonsignificant. In total, 45 of 60 propellers in the SCOP25 database, covering four SCOP folds, are clustered in this group and analysis with sensitive sequence comparison methods shows that they are similar at a level indicative of homology. Two mechanisms appear to contribute to the evolution of beta-propellers: amplification from single blades and subsequent functional differentiation. The observation of propellers with nearly identical blades in genomic sequences show that these mechanisms are still operating today.     

Structure and catalysis of acylaminoacyl peptidase: closed and open subunits of a dimer oligopeptidase

Acylaminoacyl peptidase from Aeropyrum pernix is a homodimer that belongs to the prolyl oligopeptidase family. The monomer subunit is composed of one hydrolase and one propeller domain. Previous crystal structure determinations revealed that the propeller domain obstructed the access of substrate to the active site of both subunits. Here we investigated the structure and the kinetics of two mutant enzymes in which the aspartic acid of the catalytic triad was changed to alanine or asparagine. Using different substrates, we have determined the pH dependence of specificity rate constants, the rate-limiting step of catalysis, and the binding of substrates and inhibitors. The catalysis considerably depended both on the kind of mutation and on the nature of the substrate. The results were interpreted in terms of alterations in the position of the catalytic histidine side chain as demonstrated with crystal structure determination of the native and two mutant structures (D524N and D524A). Unexpectedly, in the homodimeric structures, only one subunit displayed the closed form of the enzyme. The other subunit exhibited an open gate to the catalytic site, thus revealing the structural basis that controls the oligopeptidase activity. The open form of the native enzyme displayed the catalytic triad in a distorted, inactive state. The mutations affected the closed, active form of the enzyme, disrupting its catalytic triad. We concluded that the two forms are at equilibrium and the substrates bind by the conformational selection mechanism.     

Baicalin, a prodrug able to reach the CNS, is a prolyl oligopeptidase inhibitor

Prolyl oligopeptidase is a cytosolic serine peptidase that hydrolyzes proline-containing peptides at the carboxy terminus of proline residues. It has been associated with schizophrenia, bipolar affective disorder, and related neuropsychiatric disorders and therefore may have important clinical implications. In a previous work, we used (19)F NMR to search for new prolyl oligopeptidase inhibitors from a library of traditional Chinese medicine plant extracts, and identified several extracts as powerful inhibitors of this peptidase. Here, the flavonoid baicalin was isolated as the active component of an extract of Scutellaria baicalensis roots having prolyl oligopeptidase inhibitory activity. Baicalin inhibited prolyl oligopeptidase in a dose-dependent manner. Inhibition experiments using baicalin analogs showed that the sugar moiety was not necessary for activity. The IC(50)s of baicalin and its aglycone derivative baicalein were rather similar, showing that the sugar moiety was not involved in the interaction of baicalin with POP. These results were confirmed by saturation transfer difference NMR experiments. To further understand the absorption and transport mechanisms of baicalin and baicalein, we evaluated their transport in vitro through the gastrointestinal tract and the blood-brain barrier using a Parallel Artificial Membrane Permeability Assay. The molecule which potentially crosses both barriers was identified as baicalein, the aglycone moiety of baicalin. Our results show that baicalin is a new prodrug able to inhibit prolyl oligopeptidase. As baicalin is a natural compound with a long history of safe administration to humans, it is a highly attractive base from which to develop new treatments for schizophrenia, bipolar affective disorder, and related neuropsychiatric diseases.     

Oligopeptidase B: a processing peptidase involved in pathogenesis

Oligopeptidase B is a "processing peptidase" from the prolyl oligopeptidase family of serine peptidases present in Gram negative bacteria, protozoa and plants. Unlike the prototype prolyl oligopeptidase, oligopeptidase B hydrolyses peptides on the carboxyl side of pairs of basic amino acid residues. Molecular modelling and mutation studies have identified carboxyl dyads in the C-terminal catalytic domain that mediate substrate and inhibitor binding. The peptidase is efficiently inhibited by non-peptide irreversible serine peptidase inhibitors, peptidyl-chloromethylketones, -phosphonate alpha-aminoalkyl diphenyl esters with basic residues at P1, and tripeptide aldehydes, but not by proteinaceous host plasma inhibitors such as alpha2-macroglobulin and serpins. Access of these large molecular mass inhibitors and substrates larger than approximately 30 amino acid residues to the catalytic cleft is restricted by the N-terminal beta-propeller domain. The physiological role of oligopeptidase B from various sources has not yet been elucidated. However, the peptidase has been identified as an important virulence factor and therapeutic agent in animal trypanosomosis. This review highlights the structure-function properties of oligopeptidase B in context with its physiological and/or pathological roles which make the enzyme a promising drug target.     

FORENSIC METHODS FOR CHARACTERIZING WATERCRAFT FROM WATERCRAFT-INDUCED WOUNDS ON THE FLORIDA MANATEE (TRICHECHUS MANATUS LATIROSTRIS)

Watercraft-related mortality represents 1,253 (24.9%) of 5,033 Florida manatee ( Trichechus manatus latirostris ) deaths recorded between 1 January 1979 and 31 December 2004. Wound patterns generated by collisions with watercraft are diagnostic. Sets of cuts and scrapes that are roughly equidistant and perpendicular to the direction of vessel travel are consistent with lacerations made by propeller blades. From these lesions, estimates of propeller diameter, pitch, rotation, and direction of travel may be obtained. Considerable overlap of propeller sizes and pitches on different size vessels, common use of counter rotation propellers, and numerous other complicating factors may confound efforts to accurately predict vessel size and type from propeller wounds. Of the more than one million watercraft registered in Florida, 98% are ≤12.2 m (40 ft), yet watercraft 5.3–36.6 m (17.5–120 ft) are known to have killed manatees. Analysis of a 5-yr subset of mortality data suggests that a disproportionate number of propeller-caused watercraft-related mortalities could be attributed to propeller diameters ≥43.2 cm (17 in.), inferring that these were caused by watercraft ≥12.2 m (40 ft).     

The acylaminoacyl peptidase from Aeropyrum pernix K1 thought to be an exopeptidase displays endopeptidase activity

Mammalian acylaminoacyl peptidase, a member of the prolyl oligopeptidase family of serine peptidases, is an exopeptidase, which removes acylated amino acid residues from the N terminus of oligopeptides. We have investigated the kinetics and inhibitor binding of the orthologous acylaminoacyl peptidase from the thermophile Aeropyrum pernix K1 (ApAAP). Complex pH-rate profiles were found with charged substrates, indicating a strong electrostatic effect in the surroundings of the active site. Unexpectedly, we have found that oligopeptides can be hydrolysed beyond the N-terminal peptide bond, demonstrating that ApAAP exhibits endopeptidase activity. It was thought that the enzyme is specific for hydrophobic amino acids, in particular phenylalanine, in accord with the non-polar S1 subsite of ApAAP. However, cleavage after an Ala residue contradicted this notion and demonstrated that P1 residues of different nature may bind to the S1 subsite depending on the remaining peptide residues. The crystal structures of the complexes formed between the enzyme and product-like inhibitors identified the oxyanion-binding site unambiguously and demonstrated that the phenylalanine ring of the P1 peptide residue assumes a position different from that established in a previous study, using 4-nitrophenylphosphate. We have found that the substrate-binding site extends beyond the S2 subsite, being capable of binding peptides with a longer N terminus. The S2 subsite displays a non-polar character, which is unique among the enzymes of this family. The S3 site was identified as a hydrophobic region that does not form hydrogen bonds with the inhibitor P3 residue. The enzyme-inhibitor complexes revealed that, upon ligand-binding, the S1 subsite undergoes significant conformational changes, demonstrating the plasticity of the specificity site.     

Crystal Structures of Trypanosoma brucei Oligopeptidase B Broaden the Paradigm of Catalytic Regulation in Prolyl Oligopeptidase Family Enzymes

Oligopeptidase B cleaves after basic amino acids in peptides up to 30 residues. As a virulence factor in bacteria and trypanosomatid pathogens that is absent in higher eukaryotes, this is a promising drug target. Here we present ligand-free open state and inhibitor-bound closed state crystal structures of oligopeptidase B from Trypanosoma brucei, the causative agent of African sleeping sickness. These (and related) structures show the importance of structural dynamics, governed by a fine enthalpic and entropic balance, in substrate size selectivity and catalysis. Peptides over 30 residues cannot fit the enzyme cavity, preventing the complete domain closure required for a key propeller Asp/Glu to fix the catalytic His and Arg in the catalytically competent conformation. This size exclusion mechanism protects larger peptides and proteins from degradation. Similar bacterial prolyl endopeptidase and archael acylaminoacyl peptidase structures demonstrate this mechanism is conserved among oligopeptidase family enzymes across all three domains of life.     

Structural basis of proline-specific exopeptidase activity as observed in human dipeptidyl peptidase-IV

Inhibition of dipeptidyl peptidase IV (DPP-IV), the main glucagon-like peptide 1 (GLP1)-degrading enzyme, has been proposed for the treatment of type II diabetes. We expressed and purified the ectodomain of human DPP-IV in Pichia pastoris and determined the X-ray structure at 2.1 A resolution. The enzyme consists of two domains, the catalytic domain, with an alpha/beta hydrolase fold, and a beta propeller domain with an 8-fold repeat of a four-strand beta sheet motif. The beta propeller domain contributes two important functions to the molecule that have not been reported for such structures, an extra beta sheet motif that forms part of the dimerization interface and an additional short helix with a double Glu sequence motif. The Glu motif provides recognition and a binding site for the N terminus of the substrates, as revealed by the complex structure with diprotin A, a substrate with low turnover that is trapped in the tetrahedral intermediate of the reaction in the crystal.     

Purification and characterization of a Z-pro-prolinal-insensitive Z-Gly-Pro-7-amino-4-methyl coumarin-hydrolyzing peptidase from bovine serum--a new proline-specific peptidase.

The study of a new proline-specific peptidase from bovine serum is presented. The enzyme readily cleaves the prolyl oligopeptidase (PO) substrate Z-Gly-Pro-MCA, liberating the fluorophore MCA, thus allowing quantification of enzyme activity. Unlike PO, however, this peptidase is completely insensitive to the PO-specific inhibitor Z-Pro-prolinal and has been designated Z-Pro-prolinal-insensitive Z-Gly-Pro-MCA-hydrolyzing peptidase (ZIP). The two peptidases were successfully separated from each other by phenyl Sepharose hydrophobic interaction chromatography and the subsequent purification focused on the isolation of ZIP from bovine serum. In addition to phenyl Sepharose, calcium phosphate cellulose and DEAE anion-exchange chromatography were employed in the purification, with an overall enzyme yield of 33% and a purification factor of 4023. SDS-PAGE and size-exclusion chromatography indicated a dimeric structure with a relative molecular mass of 174 kDa. The enzyme was stable over the pH range 2.5-10.0. Optimal activity was detected in the pH range 7.4-8.0. Isoelectric focusing revealed a pI of 5.68. Inhibition by AEBSF suggests the peptidase may be a serine protease and ZIP possibly contains a cysteine residue near the active site. alpha(2)M failed to inhibit activity, suggesting oligopeptidase specificity. HPLC analysis revealed a broad substrate specificity for proline-containing peptides. Kinetic analysis indicated that ZIP had a high affinity for Z-Gly-Pro-MCA with a K(m) of 54 microM deduced. Bovine serum ZIP exhibits biophysical characteristics both similar to and different from those of PO isolated from a number of sources and may serve an important physiological function in the degradation of bioactive oligopeptides.     

Two highly conserved glutamic acid residues in the predicted beta propeller domain of dipeptidyl peptidase IV are required for its enzyme activity

Dipeptidyl peptidase IV (DPP IV) is a member of the prolyl oligopeptidase family and modifies the biological activities of certain chemokines and neuropeptides by cleaving their N-terminal dipeptides. This paper reports the identification and possible significance of a novel conserved sequence motif Asp-Trp-(Val/Ile/Leu)-Tyr-Glu-Glu-Glu (DW(V/I/L)YEEE) in the predicted beta propeller domain of the DPP IV-like gene family. Single amino acid point mutations in this motif identified two glutamates, at positions 205 and 206, as essential for the enzyme activity of human DPP IV. This observation suggests a novel role in proteolysis for residues of DPP IV distant from the Ser-Asp-His catalytic triad.     

Structure of DDB1 in complex with a paramyxovirus V protein: viral hijack of a propeller cluster in ubiquitin ligase

The DDB1-Cul4A ubiquitin ligase complex promotes protein ubiquitination in diverse cellular functions and is reprogrammed by the V proteins of paramyxoviruses to degrade STATs and block interferon signaling. Here we report the crystal structures of DDB1 alone and in complex with the simian virus 5 V protein. The DDB1 structure reveals an intertwined three-propeller cluster, which contains two tightly coupled beta propellers with a large pocket in between and a third beta propeller flexibly attached on the side. The rigid double-propeller fold of DDB1 is targeted by the viral V protein, which inserts an entire helix into the double-propeller pocket, whereas the third propeller domain docks DDB1 to the N terminus of the Cul4A scaffold. Together, these results not only provide structural insights into how the virus hijacks the DDB1-Cul4A ubiquitin ligase but also establish a structural framework for understanding the multiple functions of DDB1 in the uniquely assembled cullin-RING E3 machinery.     

Structure of the Escherichia coli TolB protein determined by MAD methods at 1.95 A resolution.

BACKGROUND: The periplasmic protein TolB from Escherichia coli is part of the Tol-PAL (peptidoglycan-associated lipoprotein) multiprotein complex used by group A colicins to penetrate and kill cells. TolB homologues are found in many gram-negative bacteria and the Tol-PAL system is thought to play a role in bacterial envelope integrity. TolB is required for lethal infection by Salmonella typhimurium in mice. RESULTS: The crystal structure of the selenomethionine-substituted TolB protein from E. coli was solved using multiwavelength anomalous dispersion methods and refined to 1. 95 A. TolB has a two-domain structure. The N-terminal domain consists of two alpha helices, a five-stranded beta-sheet floor and a long loop at the back of this floor. The C-terminal domain is a six-bladed beta propeller. The small, possibly mobile, contact area (430 A(2)) between the two domains involves residues from the two helices and the first and sixth blades of the beta propeller. All available genomic sequences were used to identify new TolB homologues in gram-negative bacteria. The TolB structure was then interpreted using the observed conservation pattern. CONCLUSIONS: The TolB beta-propeller C-terminal domain exhibits sequence similarities to numerous members of the prolyl oligopeptidase family and, to a lesser extent, to class B metallo-beta-lactamases. The alpha/beta N-terminal domain shares a structural similarity with the C-terminal domain of transfer RNA ligases. We suggest that the TolB protein might be part of a multiprotein complex involved in the recycling of peptidoglycan or in its covalent linking with lipoproteins.     

The C-terminal tails of HslU ATPase act as a molecular switch for activation of HslV peptidase

The bacterial HslVU ATP-dependent protease is a homolog of the eukaryotic 26 S proteasome. HslU ATPase forms a hexameric ring, and HslV peptidase is a dodecamer consisting of two stacked hexameric rings. In HslVU complex, the HslU and HslV central pores are aligned, and the proteolytic active sites are sequestered in an internal chamber of HslV, with access to this chamber restricted to small axial pores. Here we show that the C-terminal tails of HslU play a critical role in the interaction with and activation of HslV peptidase. A synthetic tail peptide of 10 amino acids could replace HslU in supporting the HslV-mediated hydrolysis of unfolded polypeptide substrates such as alpha-casein, as well as of small peptides, suggesting that the HslU C terminus is involved in the opening of the HslV pore for substrate entry. Moreover, deletion of 7 amino acids from the C terminus prevented the ability of HslU to form an HslVU complex with HslV. In addition, deletion of the C-terminal 10 residues prevented the formation of an HslU hexamer, indicating that the C terminus is required for HslU oligomerization. These results suggest that the HslU C-terminal tails act as a molecular switch for the assembly of HslVU complex and the activation of HslV peptidase.     

Substrate-dependent competency of the catalytic triad of prolyl oligopeptidase

Prolyl oligopeptidase, a serine peptidase unrelated to trypsin and subtilisin, is implicated in memory disorders and is an important target of drug design. The catalytic competence of the Asp(641) residue of the catalytic triad (Ser(554), Asp(641), His(680)) was studied using the D641N and D641A variants of the enzyme. Both variants displayed 3 orders of magnitude reduction in k(cat)/K(m) for benzyloxycarbonyl-Gly-Pro-2-naphthylamide. Using an octapeptide substrate, the decrease was 6 orders of magnitude, whereas with Z-Gly-Pro-4-nitrophenyl ester there was virtually no change in k(cat)/K(m). This indicates that the contribution of Asp(641) is very much dependent on the substrate-leaving group, which was not the case for the classic serine peptidase, trypsin. The rate constant for benzyloxycarbonyl-Gly-Pro-thiobenzylester conformed to this series as demonstrated by a method designed for monitoring the hydrolysis of thiolesters in the presence of thiol groups. Alkylation of His(680) with Z-Gly-Pro-CH(2)Cl was concluded with similar rate constants for wild-type and D641A variant. However, kinetic measurements with Z-Gly-Pro-OH, a product-like inhibitor, indicated that the His(680) is not accessible in the enzyme variants. Crystal structure determination of these mutants revealed subtle perturbations related to the catalytic activity. Many of these observations show differences in the catalysis between trypsin and prolyl oligopeptidase.