A doménzáródás szerepe az aktív centrum kialakításában: irányított mutagenezis vizsgálatok a 3-foszfogicerát kinázon = Role of domain closure in formation of the active site geometry by site-directed mutagenesis of 3-phosphogycerate kinase

A két doménból felépülő 3-foszfoglicerát kináz (PGK) doménzáródással működő enzim. Nem ismert, hogy mi a doménzáródás alapvető mozgató rugója, hogy miért szükséges mindkét szubsztrát egyidejű kötődése, és ez milyen molekuláris mechanizmussal indítja el a doménzáródást. Biofizikai módszerekkel és az ismert kristályszerkezetek összehasonlításával megállapítottuk, hogy miért stabilizálja bármelyik szubsztrát kötődése a másik domént és miért csak a terner komplexek esetében jön létre a zárt konformáció. A szubsztrátok együttes kötődése működteti a ?L-ben elhelyezkedő kettős molekuláris kapcsolót. A konformációváltozások közvetítésében résztvevő interdomén-régió és a MgATP kötésében résztvevő konzervatív oldalláncokat irányított mutagenezissel Ala-ra cseréltem és kinetikai ill. biofizikai vizsgálatoknak vetettem alá. Megállapításaim: 1) Az R38 mellett a K215 is katalitikus oldallánc, mely a MgATP ?-foszfátjával együtt mozdul el és azt a megfelelő helyzetbe pozícionálja a katalízis és a doménzáródás során. 2) Nukleotid szubsztrát által kiváltott konformációs hatás molekulán belüli terjedésének mechanizmusában a K219 mellett az N336 és a E343 játszik kulcs szerepet. 3) A bL redő konzervatív S392, T393, valamint a környező F165, E192 és F196 oldalláncoknak is fontos szerepe van a 3-PG indította konformációváltozás továbbításában, azonban egyetlen vizsgált oldallánc sem felelős egyedül a molekuláris csukló működtetéséért, hanem ezek együttes kapcsolatrendszere szabályozza azt. | 3-Phosphoglycerate kinase (PGK) is a two-domain hinge-bending enzyme with a well-structured interdomain region. The mechanism of domain?domain interaction, its regulation by substrate binding, the requirement of both substrates for binding and the mechanism of substrate assisted domain closure is not yet fully understood. Molecular graphical analysis of the known crystal structures and biophysical investigations have shown, that binding of either substrate stabilizes the other domain and binding of both substrates is essential for domain closure and also directs the operation of a double molecular switch at the ?L. Site directed mutations were designed at the MgATP binding site and the interdomain region: 10 conserved residues were changed to Ala and were investigated in functional and biophysical measurements. Main findings are: 1) K215 is a catalytic residue, like R38. This interacts with the g-phosphate of MgATP and assists in its proper positioning for the catalysis during domain closure. 2) The nucleotide site residues (K219, N336, E343) have essential role in the transmission of the nucleotide induced effect towards the main hinge. 3) S392 and T393 residues in bL and the surrounding F165, E192 and F196 are involved in transmission of the effects of 3-PG from one domain to the other. Neither of these side-chains is responsible alone for functioning of the molecular hinge at the ?L, rather the cumulative effects of their multiple interactions operate in the hinge region

Assessing the resilience of a river management regime: Informal learning in a shadow network in the Tisza River Basin

Global sources of change offer unprecedented challenges to conventional river management strategies, which no longer appear capable of credibly addressing a trap: the failure of conventional river defense engineering to manage rising trends of disordering extreme events, including frequency and intensity of floods, droughts, and water stagnation in the Hungarian reaches of the Tisza River Basin. Extreme events punctuate trends of stagnation or decline in the ecosystems, economies, and societies of this river basin that extend back decades, and perhaps, centuries. These trends may be the long-term results of defensive strategies of the historical river management regime that reflect a paradigm dating back to the Industrial Revolution: "Protect the Landscape from the River." Since then all policies have defaulted to the imperatives of this paradigm such that it became the convention underlying the current river management regime. As an exponent of this convention the current river management regimes' methods, concepts, infrastructure, and paradigms that reinforce one another in setting the basin's development trajectory, have proven resilient to change from wars, political, and social upheaval for centuries. Failure to address the trap makes the current river management regimes resilience appear detrimental to the regions future development prospects and prompts demand for transformation to a more adaptive river management regime. Starting before transition to democracy, a shadow network has generated multiple dialogues in Hungary, informally exploring the roots of this trap as part of a search for ideas and methods to revitalize the region. We report on how international scientists joined one dialogue, applying system dynamics modeling tools to explore barriers and bridges to transformation of the current river management regime and develop the capacity for participatory science to expand the range of perspectives that inform, monitor, and revise learning, policy, and the practice of river management

A moduláris szerveződés szerepe a fehérjék térszerkezetének kialakulásában és a katalítikus funkció megvalósításában = Role of Modular Organization in Formation of the Protein Structure and in Realization of the Enzime Function

A 3-foszfoglicerát kináz két doménje közötti együttműködés mechanizmusát írjuk le, amely a domének záródásához vezet és amely az enzim működéséhez szükséges. Funkcionális (enzimkinetika, ligandkötés, irányított mutagenézis) és szerkezeti (krisztallográfia, molekuláris grafika, modellezés, SAXS, DSC) vizsgálatainkból megállapítottuk, hogy i. a kötött szubsztrátok flexibilis foszfátjai időleges kölcsönhatásuk révén hozzájárulnak bizonyos hélixek elmozdulásához és így elősegítik a doménzáródást; ii. az R38 és K215 oldalláncok szükségesek mind a doménmozgáshoz, mind a katalízishez; iii. a terner enzim-szubsztrát komplexben kialakuló speciális H-kötés láncolat felelős az L-jelű béta-redőben lévő fő csukló régió működéséért. A termofil, mezofil és hidegtűrő izopropilmalátdehidrogenázzal végzett összehasonlító denaturációs-renaturációs vizsgálataink megmutatták, hogy a hőstabilitási különbségek a különböző denaturációs sebességekkel hozhatók összefüggésbe. A renaturációs folyamatok hasonló sebessége viszont a konzervativ oldalláncok között létrejövő specifikus kapcsolatoknak tulajdonítható. | Mechanism of interplay between two domains of 3-phosphoglycerate kinase leading to domain closure over the active site, a general problem associated with enzyme function, has been deduced from functional (enzyme kinetic, ligand binding, mutagenesis) and structural (crystallography, molecular graphics, modelling, SAXS, DSC) studies. The main points are: i. the bound substrates assist in movement of certain helices through the temporal interactions with their flexible phosphates and thereby promotes domain closure; ii. the side chains R38 and K215 are essential both in domain movement and the catalysis; iii. a special H-bond network, formed in the ternary enzyme-substrate complex, is responsible for operation of the main hinge at beta-strand L. ? Comparative unfolding-refolding studies with thermophilic, mesophilic and psychrotropic isopropylmalatedehydrogenases have revealed the importance of their different unfolding rates in their different thermal stabilities. The similar refolding rates, however, can be related to formation of specific interactions of the conserved side chains

A versatile modular vector set for optimizing protein expression among bacterial, yeast, insect and mammalian hosts

We have developed a unified, versatile vector set for expression of recombinant proteins, fit for use in any bacterial, yeast, insect or mammalian cell host. The advantage of this system is its versatility at the vector level, achieved by the introduction of a novel expression cassette. This cassette contains a unified multi-cloning site, affinity tags, protease cleavable linkers, an optional secretion signal, and common restriction endonuclease sites at key positions. This way, genes of interest and all elements of the cassette can be switched freely among the vectors, using restriction digestion and ligation without the need of polymerase chain reaction (PCR). This vector set allows rapid protein expression screening of various hosts and affinity tags. The reason behind this approach was that it is difficult to predict which expression host and which affinity tag will lead to functional expression. The new system is based on four optimized and frequently used expression systems (Escherichia coli pET, the yeast Pichia pastoris, pVL and pIEx for Spodoptera frugiperda insect cells and pLEXm based mammalian systems), which were modified as described above. The resulting vector set was named pONE series. We have successfully applied the pONE vector set for expression of the following human proteins: the tumour suppressor RASSF1A and the protein kinases Aurora A and LIMK1. Finally, we used it to express the large multidomain protein, Rho-associated protein kinase 2 (ROCK2, 164 kDa) and demonstrated that the yeast Pichia pastoris reproducibly expresses the large ROCK2 kinase with identical activity to the insect cell produced counterpart. To our knowledge this is among the largest proteins ever expressed in yeast. This demonstrates that the cost-effective yeast system can match and replace the industry-standard insect cell expression system even for large and complex mammalian proteins. These experiments demonstrate the applicability of our pONE vector set

Molecular basis for the lack of enantioselectivity of human 3-phosphoglycerate kinase

Non-natural l-nucleoside analogues are increasingly used as therapeutic agents to treat cancer and viral infections. To be active, l-nucleosides need to be phosphorylated to their respective triphosphate metabolites. This stepwise phosphorylation relies on human enzymes capable of processing l-nucleoside enantiomers. We used crystallographic analysis to reveal the molecular basis for the low enantioselectivity and the broad specificity of human 3-phosphoglycerate kinase (hPGK), an enzyme responsible for the last step of phosphorylation of many nucleotide derivatives. Based on structures of hPGK in the absence of nucleotides, and bound to l and d forms of MgADP and MgCDP, we show that a non-specific hydrophobic clamp to the nucleotide base, as well as a water-filled cavity behind it, allows high flexibility in the interaction between PGK and the bases. This, combined with the dispensability of hydrogen bonds to the sugar moiety, and ionic interactions with the phosphate groups, results in the positioning of different nucleotides so to expose their diphosphate group in a position competent for catalysis. Since the third phosphorylation step is often rate limiting, our results are expected to alleviate in silico tailoring of l-type prodrugs to assure their efficient metabolic processing

Molecular basis for the lack of enantioselectivity of human 3-phosphoglycerate kinase

Non-natural l-nucleoside analogues are increasingly used as therapeutic agents to treat cancer and viral infections. To be active, l-nucleosides need to be phosphorylated to their respective triphosphate metabolites. This stepwise phosphorylation relies on human enzymes capable of processing l-nucleoside enantiomers. We used crystallographic analysis to reveal the molecular basis for the low enantioselectivity and the broad specificity of human 3-phosphoglycerate kinase (hPGK), an enzyme responsible for the last step of phosphorylation of many nucleotide derivatives. Based on structures of hPGK in the absence of nucleotides, and bound to l and d forms of MgADP and MgCDP, we show that a non-specific hydrophobic clamp to the nucleotide base, as well as a water-filled cavity behind it, allows high flexibility in the interaction between PGK and the bases. This, combined with the dispensability of hydrogen bonds to the sugar moiety, and ionic interactions with the phosphate groups, results in the positioning of different nucleotides so to expose their diphosphate group in a position competent for catalysis. Since the third phosphorylation step is often rate limiting, our results are expected to alleviate in silico tailoring of l-type prodrugs to assure their efficient metabolic processing