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

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

Soluble components of the flagellar export apparatus, FliI, FliJ, and FliH, do not deliver flagellin, the major filament protein, from the cytosol to the export gate.

Flagella, the locomotion organelles of bacteria, extend from the cytoplasm to the cell exterior. External flagellar proteins are synthesized in the cytoplasm and exported by the flagellar type III secretion system. Soluble components of the flagellar export apparatus, FliI, FliH, and FliJ, have been implicated to carry late export substrates in complex with their cognate chaperones from the cytoplasm to the export gate. The importance of the soluble components in the delivery of the three minor late substrates FlgK, FlgL (hook-filament junction) and FliD (filament-cap) has been convincingly demonstrated, but their role in the transport of the major filament component flagellin (FliC) is still unclear. We have used continuous ATPase activity measurements and quartz crystal microbalance (QCM) studies to characterize interactions between the soluble export components and flagellin or the FliC:FliS substrate-chaperone complex. As controls, interactions between soluble export component pairs were characterized providing Kd values. FliC or FliC:FliS did not influence the ATPase activity of FliI alone or in complex with FliH and/or FliJ suggesting lack of interaction in solution. Immobilized FliI, FliH, or FliJ did not interact with FliC or FliC:FliS detected by QCM. The lack of interaction in the fluid phase between FliC or FliC:FliS and the soluble export components, in particular with the ATPase FliI, suggests that cells use different mechanisms for the export of late minor substrates, and the major substrate, FliC. It seems that the abundantly produced flagellin does not require the assistance of the soluble export components to efficiently reach the export gate

Quantification of the zymogenicity and the substrate-induced activity enhancement of complement factor D

Complement factor D (FD) is a serine protease present predominantly in the active form in circulation. It is synthesized as a zymogen (pro-FD), but it is continuously converted to FD by circulating active MASP-3. FD is a unique, self-inhibited protease. It has an extremely low activity toward free factor B (FB), while it is a highly efficient enzyme toward FB complexed with C3b (C3bB). The structural basis of this phenomenon is known; however, the rate enhancement was not yet quantified. It has also been unknown whether pro-FD has any enzymatic activity. In this study, we aimed to measure the activity of human FD and pro-FD toward uncomplexed FB and C3bB in order to quantitatively characterize the substrate-induced activity enhancement and zymogenicity of FD. Pro-FD was stabilized in the proenzyme form by replacing Arg25 (precursor numbering) with Gln (pro-FD-R/Q). Activated MASP-1 and MASP-3 catalytic fragments were also included in the study for comparison. We found that the complex formation with C3b enhanced the cleavage rate of FB by FD approximately 20 million-fold. C3bB was also a better substrate for MASP-1, approximately 100-fold, than free FB, showing that binding to C3b renders the scissile Arg-Lys bond in FB to become more accessible for proteolysis. Though easily measurable, this cleavage by MASP-1 is not relevant physiologically. Our approach provides quantitative data for the two-step mechanism characterized by the enhanced susceptibility of FB for cleavage upon complex formation with C3b and the substrate-induced activity enhancement of FD upon its binding to C3bB. Earlier MASP-3 was also implicated as a potential FB activator; however, MASP-3 does not cleave C3bB (or FB) at an appreciable rate. Finally, pro-FD cleaves C3bB at a rate that could be physiologically significant. The zymogenicity of FD is approximately 800, i.e., the cleavage rate of C3bB by pro-FD-R/Q was found to be approximately 800-fold lower than that by FD. Moreover, pro-FD-R/Q at approximately 50-fold of the physiological FD concentration could restore half-maximal AP activity of FD-depleted human serum on zymosan. The observed zymogen activity of pro-FD might be relevant in MASP-3 deficiency cases or during therapeutic MASP-3 inhibition