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

Oigopeptidázok regulációs és katalitikus mechanizmusa = Regulatory and catalytic mechanisms of oligopeptidases

Az oligopeptidázok csak kisebb peptideket hidrolizálnak. Ennek okát a proliloligopeptidáz (POP) enzimnél vizsgáltuk, amelynek fontos szerepe van a központi idegrendszer működésében. Az általunk meghatározott kristályszerkezet azt mutatta, hogy az enzim egy peptidáz és egy propeller doménből áll, és az utóbbi gátolja a nagyobb szubsztrátoknak az aktív centrumhoz való jutását. Diszulfid keresztkötésekkel és stabilitás vizsgálatokkal kimutattuk, hogy a zárt propeller nem engedheti be a szubsztrátot, az csak a két domén között, azok flexibilitása réven juthat az aktív centrumhoz. Az itt megnyíló kapu azonban kiszűri a fehérjéket. Kimutattuk továbbá, hogy a valproinsav, a depresszió egyik gyógyszere, gátolja az enzim működését. Ugyancsak megállapítottuk, hogy a POP családba tartozó PREPL fehérje, melynek hiánya súlyos betegséget okoz, nem rendelkezik hidrolitikus aktivitással. Egy másik, szintén a POP családba tartozó enzimről, az acilaminoacil peptidázról kimutattuk, hogy a katalízisben résztvevő oxianion kötőhely működését jelentősen károsítja egy a kötőhelyen kívüli aminosav mutációja. Röntgen krisztallográfiával igazoltuk, hogy a mutáció megváltoztatja a kötőhely szerkezetét. Lényeges különbséget találtunk az emlős és egy bakteriális acilaminoacil peptidáz között. Míg az emlős enzim valódi exopeptidáz, acilaminosavat hasít le a peptidlánc végéről, addig a bakteriális enzim endopeptidáz aktivitással is rendelkezik. Ez arra mutat, hogy az enzim a fejlődés során specializálódott. | Oligopeptidases hydrolyze small peptides only. The reason of the limitation was studied using prolyloligopeptidase (POP), which is involved in the function of the central nervous system. As we have shown the enzyme is composed of a peptidase and a propeller domain, the latter preventing the access of the substrate to the active site. Using disulfide cross-linking, molecular dynamics calculations and stability investigations, we have demonstrated that the substrate enters the active site between the domains, thanks to the flexibility of the protein. This gate, however, excludes the protein from the catalytic centre. We have also demonstrated that valproic acid, a drug for treating bipolar depression, inhibits POP. Furthermore, the PREPL protein of the POP family, the lack of which causes serious illness, was shown to be inactive. Acylaminoacyl peptidase, an important member of POP family, has also been investigated. A mutation outside the oxyanion binding site considerably impaired the catalytic activity due to a distortion in the structure of the oxyanion binding site, as demonstrated by X-ray crystallography. A significant difference was observed between the mammalian and a bacterial acylaminoacyl peptidase. While the mammalian enzyme proved to be a true exopeptidase cleaving acylaminoacid from the N-terminus of peptides, the bacterial enzyme also displayed endopeptidase activity, indicating that the enzyme specialized during evolution

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. © 2017 Elsevier B.V

Hipertermofil szerin oligopeptidázok szerkezete és működése = Structure and function of hyperthermophilic serine oligopeptidases

A kutatás fő célja az egyikünk által felfedezett és jellemzett prolil oligopeptidáz családhoz tartozó enzimek (prolil oligopeptidáz, acilaminoacil peptidáz) működési mechanizmusának részletes tisztázása és különböző ligandumokkal képezett komplexeinek szerkezeti elemzése volt. Az enzimek különböző variánsainak működését kombinált módszerekkel, oldatkinetikai és fehérjekrisztallográfiai vizsgálatokkal, valamint molekulamodellezéssel jellemeztük. Megállapítottuk, hogy ahhoz, hogy a katalitikus hely aktív konformációba billenjen, nem elegendőek egy kisméretű szubsztráttal kialakított nem-kovalens komplex kölcsönhatásai, hanem az enzim becsukódása és interdomén kölcsönhatásainak átrendeződése szükséges - ez biztosítja, hogy nagyobb polipeptideket és fehérjéket az enzim nem hidrolizál el. Analógiát találtunk több más fehérje (kalmodulin, dUTPáz és MASP-1), valamint az acilaminoacil peptidáz ligandumkötési mechanizmusa között. A kötőhely nyitottságának mértéke, és becsukódási képessége összefüggésbe hozható a szélesebb szelektivitással (kalmodulin és MASP-1). Az aktív hellyel közvetlenül nem érintkező konzervált aminosavak távol ható, konzervált hidrogénhíd-hálózatok révén vesznek részt a szubsztrát stabilizálásában (dUTPáz). A PCM modellel kiegészített kvantumkémiai módszerrel vizsgáltuk a fehérje gerinc proton és szén NMR kémiai eltolódásait a konformáció függvényében, és megállapítottuk, hogy a gázfázisú modellhez képest javult az egyezés a kísérleti adatokkal. | The main goal of our studies was to clarify the detailed mechanism of action of enzymes, belonging to the prolyl oligopeptidase family, discovered by one of us, as well as structural analysis of their complexes with various ligands. Activity of enzyme variants was characterised by combined methods, like solution kinetics, protein crystallography and molecular modelling. We found that, in order to switch the active site into an active conformation, it is not enough to establish non-covalent interactions with small substrates, rather closing of the enzyme and reorganisation of the interdomain interactions is needed. This ensures that larger polypeptides and proteins are not hydrolysed by the enzyme. We found an analogy among mechanisms of action of other proteins (calmodulin, dUTPase and MASP-1) and acylaminoacyl peptidase. The extent of opening at the binding site and ability to close can be related to broader selectivity (calmodulin and MASP-1). Conserved amino acid residues, not directly connected to the active site, participate in substrate stabilisation via extended, conserved H-bond networks (dUTPase). We investigated NMR proton and carbon chemical shifts of the protein backbone as a function of the conformation with a quantum chemical method extended by the PCM model and found that that the agreement with experimental data improved as compared to the gas-phase model

Properties of the prolyl oligopeptidase homologue from Pyrococcus furiosus

AbstractProlyl 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 pKa 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

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. © 2023 Elsevier B.V. and Société Française de Biochimie et Biologie Moléculaire (SFBBM