Catalytically distinct states captured in a crystal lattice: the substrate-bound and scavenger states of acylaminoacyl peptidase and their implications for functionality.

Acylaminoacyl peptidase (AAP) is an oligopeptidase that only cleaves short peptides or protein segments. In the case of AAP from Aeropyrum pernix (ApAAP), previous studies have led to a model in which the clamshell-like opening and closing of the enzyme provides the means of substrate-size selection. The closed form of the enzyme is catalytically active, while opening deactivates the catalytic triad. The crystallographic results presented here show that the open form of ApAAP is indeed functionally disabled. The obtained crystal structures also reveal that the closed form is penetrable to small ligands: inhibitor added to the pre-formed crystal was able to reach the active site of the rigidified protein, which is only possible through the narrow channel of the propeller domain. Molecular-dynamics simulations investigating the structure of the complexes formed with longer peptide substrates showed that their binding within the large crevice of the closed form of ApAAP leaves the enzyme structure unperturbed; however, their accessing the binding site seems more probable when assisted by opening of the enzyme. Thus, the open form of ApAAP corresponds to a scavenger of possible substrates, the actual cleavage of which only takes place if the enzyme is able to re-close.

A self-compartmentalizing hexamer serine protease from Pyrococcus horikoshii: substrate selection achieved through multimerization.

Oligopeptidases impose a size limitation on their substrates, the mechanism of which has long been under debate. Here we present the structure of a hexameric serine protease, an oligopeptidase from Pyrococcus horikoshii (PhAAP), revealing a complex, self-compartmentalized inner space, where substrates may access the monomer active sites passing through a double-gated "check-in" system, first passing through a pore on the hexamer surface and then turning to enter through an even smaller opening at the monomers' domain interface. This substrate screening strategy is unique within the family. We found that among oligopeptidases, a residue of the catalytic apparatus is positioned near an amylogenic ß-edge, which needs to be protected to prevent aggregation, and we found that different oligopeptidases use different strategies to achieve such an end. We propose that self-assembly within the family results in characteristically different substrate selection mechanisms coupled to different multimerization states.

The loops facing the active site of prolyl oligopeptidase are crucial components in substrate gating and specificity.

Prolyl oligopeptidase (POP) has emerged as a drug target for neurological diseases. A flexible loop structure comprising loop A (res. 189-209) and loop B (res. 577-608) at the domain interface is implicated in substrate entry to the active site. Here we determined kinetic and structural properties of POP with mutations in loop A, loop B, and in two additional flexible loops (the catalytic His loop, propeller Asp/Glu loop). POP lacking loop A proved to be an inefficient enzyme, as did POP with a mutation in loop B (T590C). Both variants displayed an altered substrate preference profile, with reduced ligand binding capacity. Conversely, the T202C mutation increased the flexibility of loop A, enhancing the catalytic efficiency beyond that of the native enzyme. The T590C mutation in loop B increased the preference for shorter peptides, indicating a role in substrate gating. Loop A and the His loop are disordered in the H680A mutant crystal structure, as seen in previous bacterial POP structures, implying coordinated structural dynamics of these loops. Unlike native POP, variants with a malfunctioning loop A were not inhibited by a 17-mer peptide that may bind non-productively to an exosite involving loop A. Biophysical studies suggest a predominantly closed resting state for POP with higher flexibility at the physiological temperature. The flexible loop A, loop B and His loop system at the active site is the main regulator of substrate gating and specificity and represents a new inhibitor target.

Probing telomeric-like G4 structures with full or partial 2'-deoxy-5-hydroxyuridine substitutions.

Guanine quadruplexes (G4s) are stable four-stranded secondary DNA structures held together by noncanonical G-G base tetrads. We synthesised the nucleoside analogue 2'-deoxy-5-hydroxyuridine (H) and inserted its phosphoramidite into telomeric repeat-type model oligonucleotides. Full and partial substitutions were made, replacing all guanines in all the three tetrads of a three-tier G4 structure, or only in the putative upper, central, or lower tetrads. We characterised these modified structures using CD, UV absorbance spectroscopy, native gel studies, and a capture oligo-based G4 disruption kinetic assay. The strand separation activity of BLM helicase on these substituted structures was also investigated. Two of the partially H-substituted constructs adopted G4-like structures, but displayed lower thermal stabilities compared to unsubstituted G4. The construct modified in its central tetrad remained mostly denatured, but the possibility of a special structure for the fully replaced variant remained open. H substitutions did not interfere with the G4-resolving activity of BLM helicase, but its efficiency was highly influenced by construct topology and even more by the G4 ligand PhenDC3. Our results suggest that the H modification can be incorporated into G quadruplexes, but only at certain positions to maintain G4 stability. The destabilizing effect observed for 2'-deoxy-5-hydroxyuridine indicates that the cytosine deamination product 5-hydroxyuracil and its nucleoside counterpart in RNA (5-hydroxyuridine), might also be destabilizing in cellular DNA and RNA quadruplexes. The kinetic assay employed in this study can be generally employed for a fast comparison of the stabilities of various G4s either in their free or ligand-bound states.

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.

BRCA1 deficiency specific base substitution mutagenesis is dependent on translesion synthesis and regulated by 53BP1.

Defects in BRCA1, BRCA2 and other genes of the homology-dependent DNA repair (HR) pathway cause an elevated rate of mutagenesis, eliciting specific mutation patterns including COSMIC signature SBS3. Using genome sequencing of knock-out cell lines we show that Y family translesion synthesis (TLS) polymerases contribute to the spontaneous generation of base substitution and short insertion/deletion mutations in BRCA1 deficient cells, and that TLS on DNA adducts is increased in BRCA1 and BRCA2 mutants. The inactivation of 53BP1 in BRCA1 mutant cells markedly reduces TLS-specific mutagenesis, and rescues the deficiency of template switch-mediated gene conversions in the immunoglobulin V locus of BRCA1 mutant chicken DT40 cells. 53BP1 also promotes TLS in human cellular extracts in vitro. Our results show that HR deficiency-specific mutagenesis is largely caused by TLS, and suggest a function for 53BP1 in regulating the choice between TLS and error-free template switching in replicative DNA damage bypass.

GAP43 shows partial co-localisation but no strong physical interaction with prolyl oligopeptidase.

It has recently been proposed that prolyl oligopeptidase (POP), the cytosolic serine peptidase with neurological implications, binds GAP43 (Growth-Associated Protein 43) and is implicated in neuronal growth cone formation, axon guidance and synaptic plasticity. We investigated the interaction between GAP43 and POP with various biophysical and biochemical methods in vitro and studied the co-localisation of the two proteins in differentiated HeLa cells. GAP43 and POP showed partial co-localisation in the cell body as well as in the potential growth cone structures. We could not detect significant binding between the recombinantly expressed POP and GAP43 using gel filtration, CD, ITC and BIACORE studies, pull-down experiments, glutaraldehyde cross-linking and limited proteolysis. However, glutaraldehyde cross-linking suggested a weak and transient interaction between the proteins. Both POP and GAP43 interacted with artificial lipids in our in vitro model system, but the presence of lipids did not evoke binding between them. In native polyacrylamide gel electrophoresis, GAP43 interacted with one of the three forms of a polyhistidine-tagged prolyl oligopeptidase. The interaction of the two proteins was also evident in ELISA and we have observed co-precipitation of the two proteins during co-incubation at higher concentrations. Our results indicate that there is no strong and direct interaction between POP and GAP43 at physiological conditions.

Evaluation and modulation of DNA lesion bypass in an SV40 large T antigen-based in vitro replication system.

DNA damage removal by nucleotide excision repair (NER) and replicative bypass via translesion synthesis (TLS) and template switch (TSw) are important in ensuring genome stability. In this study, we tested the applicability of an SV40 large T antigen-based replication system for the simultaneous examination of these damage tolerance processes. Using both Sanger and next-generation sequencing combined with lesion-specific qPCR and replication efficiency studies, we demonstrate that this system works well for studying NER and TLS, especially its one-polymerase branch, while it is less suited to investigations of homology-related repair processes, such as TSw. Cis-syn cyclobutane pyrimidine dimer photoproducts were replicated with equal efficiency to lesion-free plasmids in vitro, and the majority of TLS on this lesion could be inhibited by a peptide (PIR) specific for the polη-PCNA interaction interface. TLS on 6-4 pyrimidine-pyrimidone photoproduct proved to be inefficient and was slightly facilitated by PIR as well as by a recombinant ubiquitin-binding zinc finger domain of polη in HeLa extract, possibly by promoting polymerase exchange. Supplementation of the extract with recombinant PCNA variants indicated the dependence of TLS on PCNA ubiquitylation. In contrast to active TLS and NER, we found no evidence of successful TSw in cellular extracts. The established methods can promote in vitro investigations of replicative DNA damage bypass.

Characterization of a novel acylaminoacyl peptidase with hexameric structure and endopeptidase activity.

We have overexpressed in E. coli, purified and investigated the kinetic, thermodynamic and biophysical properties of an acylaminoacyl peptidase (AAP), from the thermophile Pyrococcus horikoshii (PhAAP). It was shown that the electrostatic environment of the catalytic site of PhAAP substantially influenced the pH dependence of the specificity rate constant (k(cat)/K(m)). However, 0.3 M NaCl, which depressed the electrostatic effects, simplified the complex pH-rate profile. The rate of formation of the enzyme-substrate complex (k(1)) was obtained from a non-linear Arrhenius plot. The lack of substrate leaving group effects indicated that k(1) is the rate determining step in the catalysis. DSC and CD measurements demonstrated that PhAAP displayed a stable structure in the catalytically competent pH range. It was shown that PhAAP is not just an acylaminoacyl peptidase, but it also has an endopeptidase activity and so differs from the mammalian AAPs. Size exclusion chromatography with PhAAP revealed a hexameric structure, which is unique among the known members of the prolyl oligopeptidase family that includes AAPs and suggests that its cellular function may be different from that of the dimeric AAP also found in the same organism.

Increase of SARS-CoV 3CL peptidase activity due to macromolecular crowding effects in the milieu composition.

The 3C-like peptidase of the severe acute respiratory syndrome virus (SARS-CoV) is strictly required for viral replication, thus being a potential target for the development of antiviral agents. In contrast to monomeric picornavirus 3C peptidases, SARS-CoV 3CLpro exists in equilibrium between the monomer and dimer forms in solution, and only the dimer is proteolytically active in dilute buffer solutions. In this study, the increase of SARS-CoV 3CLpro peptidase activity in presence of kosmotropic salts and crowding agents is described. The activation followed the Hofmeister series of anions, with two orders of magnitude enhancement in the presence of Na2SO4, whereas the crowding agents polyethylene glycol and bovine serum albumin increased the hydrolytic rate up to 3 times. Kinetic determinations of the monomer dimer dissociation constant (K(d)) indicated that activation was a result of a more active dimer, without significant changes in K(d) values. The activation was found to be independent of substrate length and was derived from both k(cat) increase and K(m) decrease. The viral peptidase activation described here could be related to the crowded intracellular environment and indicates a further fine-tuning mechanism for biological control, particularly in the microenvironment of the vesicles that are induced in host cells during positive strand RNA virus infection.

Structure, function and biological relevance of prolyl oligopeptidase.

A group of serine peptidases, the prolyl oligopeptidase family, cannot hydrolyze proteins and peptides containing more than 30 residues. The crystal structure of prolyl oligopeptidase (POP) has shown that the enzyme is composed of a peptidase domain with an alpha/beta hydrolase fold and a seven-bladed beta-propeller domain. This domain covers the catalytic triad and excludes large, structured peptides from the active site. The mechanism of substrate selection has been reviewed, along with the binding mode of the substrate and the catalytic mechanism, which differ from that of the classical serine peptidases in several features. POP is essentially a cytosolic enzyme and has been shown to be involved in a number of biological processes, but its precise function is still unknown. Many reports addressed experimentally the possible role of POP in cognitive and psychiatric processes, its involvement in the inositol phosphate signaling pathway, and its ability to metabolize bioactive peptides. Inhibitors were designed to reveal the cellular functions of POP and to treat neurological disorders. Other studies concerned the cellular localization of POP, its presumed interaction with the cytoskeletal elements, and its involvement in peptide/protein transport/secretion processes. The possible role of POP in Alzheimer disease is an intriguing issue, which is still debated. Recently, recombinant bacterial POPs have been investigated as potential therapeutics for celiac sprue, an autoimmune disease of small intestine caused by the intake of gluten proteins.

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.

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.

Fluorescence resonance energy transfer (FRET) peptides and cycloretro-inverso peptides derived from bradykinin as substrates and inhibitors of prolyl oligopeptidase.

Prolyl oligopeptidase (POP, EC 3.4.21.26) is a member of a family of serine peptidases with post-proline cleaving activity towards peptides. It is located in the cytosol in active form but without hydrolytic activity on proteins or peptides higher than 30 amino acids. Its function is not well defined, but it is involved in central nervous system disorders. Here, we studied the substrate specificity of wild type POP (POPwt) and its C255T variant lacking the non-catalytic Cys(255). This residue is located in the seven-bladed beta-propeller domain that regulates the activity of POP. Fluorescence resonance energy transfer (FRET) peptides were used with sequences derived from bradykinin-containing region of human kininogen and flanked by Abz (ortho-aminobenzoic acid) and EDDnp [N-ethylenediamine-(2,4-dinitrophenyl)]. The peptide Abz-GFSPFRQ-EDDnp was taken as leader substrate for the synthesis of five series of peptides modified at the P(3), P(2), P'(1), P'(2) and P'(3) residues. The optimal amino acids in each position for POPwt resulted in the sequence RRPYIR that is very similar to the C-terminal sequence of neurotensin. The cyclic peptides c(G((n))FSPFR) (n=1-4) were hydrolyzed by POP; their cycloretro and cycloretro-inverso analogues were inhibitors in the micromolar range. The differences between POPwt and its C255T mutant in the hydrolysis of the series derived from Abz-GFSPFRQ-EDDnp were restricted to the non-prime site of the substrates. The kinetic data of hydrolysis and inhibition by the cyclic peptides are consistent with the structures of POP-substrate/inhibitor complexes and with the substrate specificity data obtained with linear FRET peptides. All together, these results give information about the POP-substrate/inhibitor interactions that further complete knowledge of this important oligopeptidase.

A genetic study based on PCNA-ubiquitin fusions reveals no requirement for PCNA polyubiquitylation in DNA damage tolerance.

Post-translational modifications of Proliferating Cell Nuclear Antigen (PCNA) play a key role in regulating the bypass of DNA lesions during DNA replication. PCNA can be monoubiquitylated at lysine 164 by the RAD6-RAD18 ubiquitin ligase complex. Through this modification, PCNA can interact with low fidelity Y family DNA polymerases to promote translesion synthesis. Monoubiquitylated PCNA can be polyubiquitylated on lysine 63 of ubiquitin by a further ubiquitin-conjugating complex. This modification promotes a template switching bypass process in yeast, while its role in higher eukaryotes is less clear. We investigated the function of PCNA ubiquitylation using a PCNAK164R mutant DT40 chicken B lymphoblastoma cell line, which is hypersensitive to DNA damaging agents such as methyl methanesulfonate (MMS), cisplatin or ultraviolet radiation (UV) due to the loss of PCNA modifications. In the PCNAK164R mutant we also detected cell cycle arrest following UV treatment, a reduced rate of damage bypass through translesion DNA synthesis on synthetic UV photoproducts, and an increased rate of genomic mutagenesis following MMS treatment. PCNA-ubiquitin fusion proteins have been reported to mimic endogenous PCNA ubiquitylation. We found that the stable expression of a PCNAK164R-ubiquitin fusion protein fully or partially rescued the observed defects of the PCNAK164R mutant. The expression of a PCNAK164R-ubiquitinK63R fusion protein, on which the formation of lysine 63-linked polyubiquitin chains is not possible, similarly rescued the cell cycle arrest, DNA damage sensitivity, reduction of translesion synthesis and increase of MMS-induced genomic mutagenesis. Template switching bypass was not affected by the genetic elimination of PCNA polyubiquitylation, but it was reduced in the absence of the recombination proteins BRCA1 or XRCC3. Our study found no requirement for PCNA polyubiquitylation to protect cells from replication-stalling DNA damage.

Properties of the prolyl oligopeptidase homologue from Pyrococcus furiosus.

Prolyl oligopeptidase (POP), the paradigm of a serine peptidase family, hydrolyses peptides, but not proteins. The thermophilic POP from Pyrococcus furiosus (Pfu) appeared to be an exception, since it hydrolysed large proteins. Here we demonstrate that the Pfu POP does not display appreciable activity against azocasein. The autolysis observed earlier was an artefact. We have also found that the pH-rate profile is different from that of the mammalian enzyme and the low pK(a) extracted from the curve represents the ionization of the catalytic histidine. We conclude that some oligopeptidases may be true endopeptidases, cleaving at disordered segments of proteins, but with very low efficacy.

Unclosed beta-propellers display stable structures: implications for substrate access to the active site of prolyl oligopeptidase.

Prolyl oligopeptidase is implicated in the metabolism of neuropeptides and is involved in amnesia and depression. It contains a peptidase and an unusual beta-propeller domain that excludes large peptides and proteins from the active site. The propeller consists of seven blades not closed by a "Velcro" between the first and last blades. The propeller domain was expressed as a stable, soluble protein, P(7). Its conformational identity with that of the native propeller was verified by circular dichroism and digestion with trypsin. Differential scanning calorimetry, kinetic denaturation with urea and equilibrium denaturation with guanidinium chloride have shown that the propeller is more stable than the parent prolyl oligopeptidase. The deletion of the seventh blade of P(7) led to a stable structure, a six-bladed propeller, P(6), which immediately dimerized, in contrast with the monomeric P(7). Addition of an 11 amino acid residue extension to the C terminus of P(6) also produced a dimer, whereas the P(6) labelled with a His-tag at the N terminus displayed a monomer structure. The stability of P(6) and its variants was lower than that of P(7). The denatured propellers refolded readily. This study shows that the unclosed P(7) is a stable structure, and suggests that an opening between the peptidase and the propeller domains is more important for the substrate entry than is the putative opening between the first and seventh blades. Our results suggest that the propellers are simple, versatile structures, which can be prepared artificially.

A comprehensive survey of the mutagenic impact of common cancer cytotoxics.

BACKGROUND: Genomic mutations caused by cytotoxic agents used in cancer chemotherapy may cause secondary malignancies as well as contribute to the evolution of treatment-resistant tumour cells. The stable diploid genome of the chicken DT40 lymphoblast cell line, an established DNA repair model system, is well suited to accurately assay genomic mutations. RESULTS: We use whole genome sequencing of multiple DT40 clones to determine the mutagenic effect of eight common cytotoxics used for the treatment of millions of patients worldwide. We determine the spontaneous mutagenesis rate at 2.3 × 10(-10) per base per cell division and find that cisplatin, cyclophosphamide and etoposide induce extra base substitutions with distinct spectra. After four cycles of exposure, cisplatin induces 0.8 mutations per Mb, equivalent to the median mutational burden in common leukaemias. Cisplatin-induced mutations, including short insertions and deletions, are mainly located at sites of putative intrastrand crosslinks. We find two of the newly defined cisplatin-specific mutation types as causes of the reversion of BRCA2 mutations in emerging cisplatin-resistant tumours or cell clones. Gemcitabine, 5-fluorouracil, hydroxyurea, doxorubicin and paclitaxel have no measurable mutagenic effect. The cisplatin-induced mutation spectrum shows good correlation with cancer mutation signatures attributed to smoking and other sources of guanine-directed base damage. CONCLUSION: This study provides support for the use of cell line mutagenesis assays to validate or predict the mutagenic effect of environmental and iatrogenic exposures. Our results suggest genetic reversion due to cisplatin-induced mutations as a distinct mechanism for developing resistance.

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.

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.