A fehérjék világa | Proteins – From Structure to Function, from Physics to Biology

Amikor az élővilág bonyolult és sokszor csodálatos jelenségeivel nap mint nap találkozunk, ritkán gondolunk arra, hogy egy termést hozó fa, egy ásító oroszlán vagy egy tervezőmérnök tevékenységének hátterében egy rendkívül bonyolult mikrovilágnak – a fehérjék világának – összehangolt működése rejlik. Szervezetünk építőkövei, anyagcserénk katalizátorai, egészségünk védelmezői, energiaellátásunk szervezői, tagjaink mozgatói – mind-mind fehérjemolekulák. A fehérjék hasonló atomokból, ugyanolyan fizikai kölcsönhatások szerint épülnek fel, mint egy ásványdarab vagy egy nejlonharisnya. A különbség a célszerűen „tervezett” térszerkezet eredménye. Az előadás a fehérjék mikrovilágába vezet el bennünket, megmutatja atomi szintű szerkezetüket, és levezeti ebből azt a csodálatosan komplex jelenséget, amit életnek nevezünk. | When we admire the wonders of the living world, we rarely think about proteins. However, behind the movement of our muscles, the division of our cells, our thoughts, behind the complex phenomena of life in general, we can discover the concerted action of macromolecules - first of all proteins. The amount of our knowledge about the structure and function of proteins is immense, but far not complete. Proteins and dead matter are composed of similar atoms and are stabilized by the same physical forces. The lecture gives us an insight into the physical background of the sophisticated spatial structure of protein molecules, and into the delicately tuned and regulated function of enzymes. The principles and mechanisms of folding, assembly and self organization of proteins and their complexes are discussed. Finally the lecture touches upon the fact that a profound understanding of proteins also requires the application of the concept of quantum

Az MBL-hez kapcsolódó szerin proteázok szubsztrát specificitása és fiziológiai jelentősége = Substrate specificity and physiological relevance of MBL-associated serine proteases

A komplement rendszer aktiválódásának lektin útja az egyik első védelmi vonalnak tekinthető a szervezet fertőzések elleni védekezésében. A mannóz kötő lektin (MBL) baktérium felszínhez való kötődése után szerin proteáz zimogének (MASP= MBL-kötött szerin proteáz) aktiválódnak, melyek többféle mechanizmus révén járulnak hozzá az idegen mikroorganizmus megsemmisítéséhez ill. eltávolításához. Munkánk során felderítettük, a proteolitikus kaszkádrendszer beindításáért felelős MASP-2 enzim autoaktiválódásásnak mechanizmusát atomi szinten. Felfedeztük a MASP-2 egy eddig ismeretlen biológiai funkcióját, amely kapcsolatot teremt a véralvadási és a komplement kaszkád között. A MASP-2 hasítja és aktiválja a protrombint. Ugyancsak részletesen tanulmányoztuk a MASP-1 trombin-szerű aktivitását is. Ezek az eredmények arra utalnak, hogy a vérben lévő két proteolitikus kaszkádrendszer szoros evolúciós és funkcionális kapcsolatban van egymással, a komplement lektin útja által indukált limitált koaguláció az immunvédekezés egy ősi formájának tekinthető. | The lectin pathway of the complement system forms one of the first defence lines against the infections in our body. Upon MBL (mannose-binding lectin) binds to the bacterial surface serine protease zymogens (MASP=MBL-associated serine protease) become activated, and the active MASPs contribute to the inactivation and elimination of the foreign microorganism in several ways. In the course of our work we revealed the detailed atomic mechanism of the autoactivation of MASP-2 that is responsible for the initiation of the complement cascade. We discovered a new biological function of MASP-2 which makes contact between the complement and the coagulation cascades. MASP-2 cleaves and activates prothrombin. We also studied the thrombin-like activity of MASP-1 in detail. These results suggest that the two proteolytic cascade systems are in close evolutionary and functional relationship. The limited coagulation induced by the lectin pathway of the complement system can be regarded as an ancient form of immunity

Calcium-dependent conformational flexibility of a CUB domain controls activation of the complement serine protease C1r.

C1, the first component of the complement system, is a Ca(2+)-dependent heteropentamer complex of C1q and two modular serine proteases, C1r and C1s. Current functional models assume significant flexibility of the subcomponents. Noncatalytic modules in C1r have been proposed to provide the flexibility required for function. Using a recombinant CUB2-CCP1 domain pair and the individual CCP1 module, we showed that binding of Ca(2+) induces the folding of the CUB2 domain and stabilizes its structure. In the presence of Ca(2+), CUB2 shows a compact, folded structure, whereas in the absence of Ca(2+), it has a flexible, disordered conformation. CCP1 module is Ca(2+)-insensitive. Isothermal titration calorimetry revealed that CUB2 binds a single Ca(2+) with a relatively high K(D) (430 mum). In blood, the CUB2 domain of C1r is only partially (74%) saturated by Ca(2+), therefore the disordered, Ca(2+)-free form could provide the flexibility required for C1 activation. In accordance with this assumption, the effect of Ca(2+) on the autoactivation of native, isolated C1r zymogen was proved. In the case of infection-inflammation when the local Ca(2+) concentration decreases, this property of CUB2 domain could serve as subtle means to trigger the activation of the classical pathway of complement. The CUB2 domain of C1r is a novel example for globular protein domains with marginal stability, high conformational flexibility, and proteolytic sensitivity. The physical nature of the behavior of this domain is similar to that of intrinsically unstructured proteins, providing a further example of functionally relevant ligand-induced reorganization of a polypeptide chain

Extensive Basal Level Activation of Complement Mannose-Binding Lectin-Associated Serine Protease-3: Kinetic Modeling of Lectin Pathway Activation Provides Possible Mechanism

Serine proteases (SPs) are typically synthesized as precursors, termed proenzymes or zymogens, and the fully active form is produced via limited proteolysis by another protease or by autoactivation. The lectin pathway of the complement system is initiated by mannose-binding lectin (MBL)-associated SPs (MASP)-1, and MASP-2, which are known to be present as proenzymes in blood. The third SP of the lectin pathway, MASP-3, was recently shown to be the major activator, and the exclusive "resting blood" activator of profactor D, producing factor D, the initiator protease of the alternative pathway. Because only activated MASP-3 is capable of carrying out this cleavage, it was presumed that a significant fraction of MASP-3 must be present in the active form in resting blood. Here, we aimed to detect active MASP-3 in the blood by a more direct technique and to quantitate the active to zymogen ratio. First, MASPs were partially purified (enriched) from human plasma samples by affinity chromatography using immobilized MBL in the presence of inhibitors. Using this MASP pool, only the zymogen form of MASP-1 was detected by Western blot, whereas over 70% MASP-3 was in an activated form in the same samples. Furthermore, the active to zymogen ratio of MASP-3 showed little individual variation. It is enigmatic how MASP-3, which is not able to autoactivate, is present mostly as an active enzyme, whereas MASP-1, which has a potent autoactivation capability, is predominantly proenzymic in resting blood. In an attempt to explain this phenomenon, we modeled the basal level fluid-phase activation of lectin pathway proteases and their subsequent inactivation by C1 inhibitor and antithrombin using available and newly determined kinetic constants. The model can explain extensive MASP-3 activation only if we assume efficient intracomplex activation of MASP-3 by zymogen MASP-1. On the other hand, the model is in good agreement with the fact that MASP-1 and -2 are predominantly proenzymic and some of them is present in the form of inactive serpin-protease complexes. As an alternative hypothesis, MASP-3 activation by proprotein convertases is also discussed

Szerin proteázok az immunrendszerben: szerkezet, funkció, fiziológiai jelentőség = Serine proteases of the immune system: structure, function, physiological significance

A komplement rendszer egy szerin proteázokból álló kaszkádrendszer, amely a természetes immunitás részeként fontos szerepet tölt be a fertőzések elleni védekezésben. Munkánk során tanulmányoztuk a komplement aktiválódás klasszikus és lektin útjában résztvevő szerin proteáz enzimek szerkezetét, működését és fiziológiai jelentőségét. Meghatároztuk a lektin út kulcsenzimének, a MASP-2-nek a térszerkezetét aktív és zimogén formában. Jellemeztük a két forma enzimatikus tulajdonságait is. A szerkezetekből és mérésekből kiindulva modelleztük a MASP-2 szubsztrát-indukálta autoaktiválódási mechanizmusát atomi szinten. Ugyancsak meghatároztuk a C1r, a klasszikus aktiválódási út során autoaktiválódó, szerin proteáz térszerkezetét. A térszekezet és mérési adatok alapján egy új modellt alkottunk a klasszikus út iniciációs lépésére, a C1r autokativálódására a C1 komplexen belül. NMR spektroszkópia segítségével vizsgáltuk, hogy a C1r molekula mely szegmense felelős a funkcióhoz szükséges konformációs flexibilitásért. A komplement proteázok szubsztrátspecificitásának tanulmányozása során megerősítettük, hogy a vérben található két proteolitikus kaszkádrendszer, a véralvadás és a komplement, nem függetlenek egymástól, hanem több ponton is kölcsönhatásban állnak. Sikerült meghatároznunk a korai komplement proteázok közös fiziológiás inhibitorának, a C1-inhibitornak a térszerkezetét. A térszerkezet alapján megmagyaráztuk a heparin inhibitor-moduláló hatását, valamint a betegséget (örökletes angioödéma) okozó mutációk szerkezeti hátterét. | The complement system is a proteolytic cascade system that plays an essential role in the innate immunity. We studied the structure, function and physiological relevance of the serine proteases involved in the classical and lectin activation pathways of complement. We determined the 3D structures of the zymogen and active forms MASP-2 which is the key enzyme of the lectin pathway. We also characterized the enzymatic properties of the two forms. Using the structural and experimental data we built an in silico model for the mechanism of the substrate-induced autoactivation of MASP-2. We also solved the crystal structure of Cr, which is the autoactivating protease of the classical pathway. Based on our data we built a new functional model for the initiation step of the classical pathway: the autoactivation of C1r in the C1 complex. We used NMR to study the conformational flexibility of C1r in solution. Our study concerning the substrate specificity of the complement proteases reinforced our view that the two proteolytic cascade systems that are present in the blood plasma (ie the complement and the coagulation systems) are not independent, but interact with each-other in several ways. We managed to determine the 3D structure of the C1-inhibitor, which is the common inhibitor of the early complement proteases and some other proteases in the blood. The structure revealed the background of the inhibitor function modulating effect of heparin and we could explain the structural basis of some mutation which lead to the development of the disease, hereditary angioedema

Összefüggés a fehérjék stabilitása, konformációs flexibilitása és működése között = Relationship between stability, conformational flexibility and function of proteins

Homológ hőkedvelő, mezofil és hidegtűrő enzimek sorozatainak szerkezeti, szerveződési (folding) és funkcionális (reakció kinetikai) összehasonlító vizsgálatát végeztük el. A katalitikus reakció enzimkinetikai paramétereinek a hőmérséklet és a konformációs fluktuációk (relaxációs spektrum) függvényében történő analízisével megállapítottuk, hogy a gliceraldehid-3-foszfát dehidrogenáz enzim alegységei közötti allosztérikus jeltovábbításban a molekula dinamikus tulajdonságainak meghatározó szerepe van. A több doménből álló modellfehérjék (IPMDH, PGK) esetében feltártuk a doménzáródás atomi szintű lépéseinek összefüggését a katalitikus folyamat egyes elemeivel. A fehérjék folding-refolding folyamatainak kinetikai analízísével több, a hőstabilitás mechanizmusát értelmező új megállapítást tettünk. Vizsgáltuk a Thermotoga maritima eubaktériumból származó, hasonlóságot mutató termofil és hipertermofil xilanázok hőstabilitása közötti különbség szerkezeti hátterét. Irányított family shuffling segítségével számos kiméra enzimet állítottunk elő a két vad típusú xilanázból. A termofil xilanáz enzim terminális régióit lecserélve a hipertermofil xilanáz enzim terminális régióira, a termofil enzim hőstabilitását 12 oC-kal megnöveltük. A létrehozott kiméra konstrukciók arra világítottak rá, hogy a terminális régiók közötti kölcsönhatás stabilizálásával megnövelhető azon fehérjék hőstabilitása, amelyek terminális régiói megfelelő közelségben találhatóak. | Comparative structural, folding and functional studies were completed using sets of homologous thermophilic, mesophilic, and psychrotropic enzymes. It was revealed that the dynamic features of the enzyme molecule are essential for the mechanism of intramolecular, allosteric signal transduction between subunits of glyceraldehyde-3-phosphate dehydrogenase. The steps of domain closure, at the atomic level, were related to individual elements of the catalytic process in the case of multidomain enzymes (IPMDH, PGK). A novel mechanistic interpretation was developed based on kinetic analyses of folding-refolding processes. The structural background of the difference in heat stabilities between homologous thermophilic and hyperthermophilic xylanases from Thermotoga maritima was studied. Several chimeric constructs of the two parental enzymes were produced by directed family shuffling. A significant increase in heat stability was achieved in the case of one construct. Exchanging the terminal regions of the thermophilic enzyme by the respective fragments of the hyperthermophilic xylanase, the heat stability of the enzyme increased by 12 oC. Characterization of chimeric constructs showed that the interactions between terminal regions have an important role in stabilizing protein structure, and based on this observation, heat stability can be increased in proteins in which the terminal regions are in close proximity

MASP-1 and MASP-2 Do Not Activate Pro-Factor D in Resting Human Blood, whereas MASP-3 Is a Potential Activator: Kinetic Analysis Involving Specific MASP-1 and MASP-2 Inhibitors.

It had been thought that complement factor D (FD) is activated at the site of synthesis, and only FD lacking a propeptide is present in blood. The serum of mannose-binding lectin-associated serine protease (MASP)-1/3(-/-) mice contains pro-FD and has markedly reduced alternative pathway activity. It was suggested that MASP-1 and MASP-3 directly activate pro-FD; however, other experiments contradicted this view. We decided to clarify the involvement of MASPs in pro-FD activation in normal, as opposed to deficient, human plasma and serum. Human pro-FD containing an APPRGR propeptide was produced in insect cells. We measured its activation kinetics using purified active MASP-1, MASP-2, MASP-3, as well as thrombin. We found all these enzymes to be efficient activators, whereas MASP proenzymes lacked such activity. Pro-FD cleavage in serum or plasma was quantified by a novel assay using fluorescently labeled pro-FD. Labeled pro-FD was processed with t1/2s of approximately 3 and 5 h in serum and plasma, respectively, showing that proteolytic activity capable of activating pro-FD exists in blood even in the absence of active coagulation enzymes. Our previously developed selective MASP-1 and MASP-2 inhibitors did not reduce pro-FD activation at reasonable concentration. In contrast, at very high concentration, the MASP-2 inhibitor, which is also a poor MASP-3 inhibitor, slowed down the activation. When recombinant MASPs were added to plasma, only MASP-3 could reduce the half-life of pro-FD. Combining our quantitative data, MASP-1 and MASP-2 can be ruled out as direct pro-FD activators in resting blood; however, active MASP-3 is a very likely physiological activator

Engineering the thermostability of a TIM-barrel enzyme by rational family shuffling

a b s t r a c t A possible approach to generate enzymes with an engineered temperature optimum is to create chimeras of homologous enzymes with different temperature optima. We tested this approach using two family-10 xylanases from Thermotoga maritima: the thermophilic xylanase A catalytic domain (TmxAcat, T opt = 68°C), and the hyperthermophilic xylanase B (TmxB, T opt = 102°C). Twenty-one different chimeric constructs were created by mimicking family shuffling in a rational manner. The measured temperature optima of the 16 enzymatically active chimeras do not monotonically increase with the percentage of residues coming from TmxB. Only four chimeras had a higher temperature optimum than TmxAcat, the most stable variant (T opt = 80°C) being the one in which both terminal segments came from TmxB. Further analysis suggests that the interaction between the N-and C-terminal segments has a disproportionately high contribution to the overall thermostability. The results may be generalizable to other enzymes where the N-and C-termini are in contact. Ó 2008 Elsevier Inc. All rights reserved. Microorganisms occur in almost all environments on Earth, including high-temperature environments such as hot springs. In most cases, proteins from (hyper)thermophilic organisms have been found to be structurally similar to their mesophilic counterparts, except for minor differences The (b/a) 8 -barrel fold, which was found in triose-phosphate isomerase, and is therefore also known as the TIM-barrel fold, is the most common enzyme fold In the present study, we used two family-10 xylanases from the hyperthermophilic eubacterium Thermotoga maritima MSB8 with widely different temperature optima as starting points for rational family shuffling Materials and methods Construction of chimeric enzymes. Genes xynAcat (GenBank Accession No. Z46264, basepairs 1340-2323) and xynB (GenBank Accession No. AAD35164) encoding TmxB and TmxAcat were PCR 0006-291X/$ -see front matter