Publikációk

2012

Zoltan Ujfalusi, Mihaly Kovacs, Nikolett T Nagy, Szilvia Barko, Gabor Hild, Andras Lukacs, Miklos Nyitrai, Beata Bugyi

Myosin and tropomyosin stabilize the conformation of formin-nucleated actin filaments

JOURNAL OF BIOLOGICAL CHEMISTRY 287

The conformational elasticity of the actin cytoskeleton is essential for its versatile biological functions. Increasing evidence supports that the interplay between the structural and functional properties of actin filaments is finely regulated by actin-binding proteins; however, the underlying mechanisms and biological consequences are not completely understood. Previous studies showed that the binding of formins to the barbed end induces conformational transitions in actin filaments by making them more flexible through long range allosteric interactions. These conformational changes are accompanied by altered functional properties of the filaments. To get insight into the conformational regulation of formin-nucleated actin structures, in the present work we investigated in detail how binding partners of formin-generated actin structures, myosin and tropomyosin, affect the conformation of the formin-nucleated actin filaments using fluorescence spectroscopic approaches. Time-dependent fluorescence anisotropy and temperature-dependent Förster-type resonance energy transfer measurements revealed that heavy meromyosin, similarly to tropomyosin, restores the formin-induced effects and stabilizes the conformation of actin filaments. The stabilizing effect of heavy meromyosin is cooperative. The kinetic analysis revealed that despite the qualitatively similar effects of heavy meromyosin and tropomyosin on the conformational dynamics of actin filaments the mechanisms of the conformational transition are different for the two proteins. Heavy meromyosin stabilizes the formin-nucleated actin filaments in an apparently single step reaction upon binding, whereas the stabilization by tropomyosin occurs after complex formation. These observations support the idea that actin-binding proteins are key elements of the molecular mechanisms that regulate the conformational and functional diversity of actin filaments in living cells.

Ma X, Kovaćs M, Conti M A, Wang A, Zhang Y, Sellers J R, Adelstein R S

Nonmuscle myosin II exerts tension but does not translocate actin in vertebrate cytokinesis

PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA 109

During vertebrate cytokinesis it is thought that contractile ring constriction is driven by nonmuscle myosin II (NM II) translocation of antiparallel actin filaments. Here we report in situ, in vitro, and in vivo observations that challenge this hypothesis. Graded knockdown of NM II in cultured COS-7 cells reveals that the amount of NM II limits ring constriction. Restoration of the constriction rate with motor-impaired NM II mutants shows that the ability of NM II to translocate actin is not required for cytokinesis. Blebbistatin inhibition of cytokinesis indicates the importance of myosin strongly binding to actin and exerting tension during cytokinesis. This role is substantiated by transient kinetic experiments showing that the load-dependent mechanochemical properties of mutant NM II support efficient tension maintenance despite the inability to translocate actin. Under loaded conditions, mutant NM II exhibits a prolonged actin attachment in which a single mechanoenzymatic cycle spans most of the time of cytokinesis. This prolonged attachment promotes simultaneous binding of NM II heads to actin, thereby increasing tension and resisting expansion of the ring. The detachment of mutant NM II heads from actin is enhanced by assisting loads, which prevent mutant NM II from hampering furrow ingression during cytokinesis. In the 3D context of mouse hearts, mutant NM II-B R709C that cannot translocate actin filaments can rescue multinucleation in NM II-B ablated cardiomyocytes. We propose that the major roles of NM II in vertebrate cell cytokinesis are to bind and cross-link actin filaments and to exert tension on actin during contractile ring constriction.

Lada Biedermannova, Zbynek Prokop, Artur Gora, Eva Chovancova, Mihaly Kovacs, Jiri Damborsky, Rebecca C Wade

A single mutation in a tunnel to the active site changes the mechanism and kinetics of product release in haloalkane dehalogenase LinB

JOURNAL OF BIOLOGICAL CHEMISTRY 287

Many enzymes have buried active sites. The properties of the tunnels connecting the active site with bulk solvent affect ligand binding and unbinding and also the catalytic properties. Here, we investigate ligand passage in the haloalkane dehalogenase enzyme LinB and the effect of replacing leucine by a bulky tryptophan at a tunnel-lining position. Transient kinetic experiments show that the mutation significantly slows down the rate of product release. Moreover, the mechanism of bromide ion release is changed from a one-step process in the wild type enzyme to a two-step process in the mutant. The rate constant of bromide ion release corresponds to the overall steady-state turnover rate constant, suggesting that product release became the rate-limiting step of catalysis in the mutant. We explain the experimental findings by investigating the molecular details of the process computationally. Analysis of trajectories from molecular dynamics simulations with a tunnel detection software reveals differences in the tunnels available for ligand egress. Corresponding differences are seen in simulations of product egress using a specialized enhanced sampling technique. The differences in the free energy barriers for egress of a bromide ion obtained using potential of mean force calculations are in good agreement with the differences in rates obtained from the transient kinetic experiments. Interactions of the bromide ion with the introduced tryptophan are shown to affect the free energy barrier for its passage. The study demonstrates how the mechanism of an enzymatic catalytic cycle and reaction kinetics can be engineered by modification of protein tunnels.

Harami Gábor, Gyimesi Máté, Kovács Mihály

A kulcstól a bulldózerig: szárnyas hélix domének funkcionális adaptációja DNS-kötő fehérjékben

BIOKÉMIA 36/2

Motorenzimológiai kutatócsoportunk egyik fő érdeklődési területe a RecQ családba tartozó DNS-helikázok molekuláris működése és ennek szerepe a genomkarbantartó folyamatokban. A RecQ helikázok nélkülözhetetlen szereplői a DNS-hibák homológ rekombináción (HR) alapuló javításának. E működés alapfeltétele a RecQ helikázok összetett DNS-szerkezetek felismerésére és feldolgozására való képessége. A felismerés és feldolgozás mechanizmusa és szerkezeti alapjai azonban az intenzív vizsgálatok ellenére nagyrészt felderítetlenek. Az eddigi eredmények alapján úgy tűnik, hogy a sokféle DNS-kötő fehérjében - így a RecQ helikázokban is - megtalálható szárnyas hélix (winged helix, WH) domén kulcsszereplője a DNS-szerkezetek felismerésének. E domén több száz fehérje - főként transzkripciós faktorok - esetében szekvencia-specifikus kettősszálú (double-stranded, ds) DNS-kötést („kulcs”-funkciót) tesz lehetővé. Közelmúltbeli felfedezések azonban azt sugallják, hogy a WH domén a RecQ helikázokban különböző DNS-száltalálkozások és szálszerkezetek szekvencia-aspecificikus felismerésére és szétválasztására („bulldózer”- funkcióra) adaptálódott. Jelen összefoglalónkban a WH domének szerkezeti sajátosságait a RecQ helikázokban betöltött, mások és magunk által felderített funkcióival összekapcsolva mutatjuk be.

Kiss Bence,Nyitray László

A KUPLUNG FELENGEDÉSE: AZ S100A4 - MIOZIN IIA KÖLCSÖNHATÁS ÉS A SEJTMIGRÁCIÓ

BIOKÉMIA 36/2

Az S100A4 (metasztazin) egy, a Ca2+-kötő S100 fehérjecsaládba tartozó, gerinces-specifikus homodimer fehérje, mely összefüggésbe hozható számos ráktípus áttétképző hajlamával és több más, megnövekedett sejtmigrációval járó betegséggel. Ca2+-függő módon kötődik a nem-izom sejtekre jellemző miozin IIA (NMIIA) motorfehérjéhez. A kölcsönhatás következtében a miozin filamentumok depolimerizálnak, a sejtek motilitása fokozódik. Meghatároztuk az S100A4 - NMIIA komplex atomi felbontású szerkezetét, és mechanisztikus magyarázatot adunk a filamentumok szétesésének molekuláris hátterére. A három gerinces NMII izoforma kötési szelektivitását vizsgálva kimutattuk, hogy az S100A4 az NMIIAhoz hasonló affinitással kötődik az NMIIC izoformához is, amely tény segíthet az NMIIC eddig kevéssé ismert in vivo szerepének feltárásában. Az S100A4 - NMIIA komplex szerkezete merőben új típusú, aszimmetrikus kölcsönhatásra világít rá az S100 családban: egyetlen, zömében .-helikális miozin peptid körülöleli az S100A4 homodimert, elfoglalván mindkét „kanonikus” kötőzsebet. Kísérleteink alapján a filamentumok depolimerizációja a következő mechanizmussal megy végbe: az S100A4 dimerek megkötik a miozin nehézlánc C-terminális random-coil régióját, majd magukra tekerik a szomszédos coiled-coil régiót. Ezzel tönkreteszik a dimerizációért és a filamentumok kialakulásáért felelős ún. assembly competence domént, a miozin filamentum szétesésnek, így a retrográd aktin áramlásért felelős miozin gátlódik, s az aktin polimerizáció erőhatása a sejtet előre tudja mozgatni. Nem kerülte el a figyelmünket, hogy a komplex szerkezetének ismerete elősegítheti terápiás célú S100A4 inhibitorok fejlesztését is.

Simon Zoltán,Peragovics Ágnes,Málnási-Csizmadia András

Gyógyszerprofil-összevetés: a polifarmakológia lehetőségei

BIOKÉMIA 36/2

A gyógyszerek és gyógyszerjelöltek teljes hatásprofiljának előrejelzése eddig nem volt lehetséges. A cikkben bemutatott Drug Profile Matching (Gyógyszerprofil-összevetés, DPM) módszer alapját az a hipotézis képezi, hogy komplex molekuláris tulajdonságkészletek, mint amilyen egy kötési mintázat, korrelációt mutatnak a hatásprofilok ismert elemeivel, és ezáltal prediktív erővel rendelkeznek a teljes profil előállítása felé. Módszerünk nem vizsgálja a kismolekulák kémiai hasonlóságát. Ehelyett olyan kölcsönhatási eredményekre épül, melyek kísérletesen nem, vagy csak nehézkesen mutathatók ki, mivel nem célfehérjéhez történő kölcsönhatásokat írnak le. A DPM gyors és szisztematikus eljárás, mellyel ismert gyógyszerek új indikációi és biztonsági kockázatai deríthetők fel, továbbá alkalmazható a gyógyszerjelöltek hatásprofiljának predikciójára, s ezáltal a vezérmolekula-tervezés és optimalizáció terén is hatékony eszköz lehet. Ezzel segítséget nyújt például új antipszichotikumok fejlesztésében: a módszer segítségével olyan újrapozícionálási lehetőségekre derülhet fény, melyek hagyományos keresési eljárásokkal nem kerülnének előtérbe - nem beszélve a kémiailag új gyógyszerjelöltekről, melyek nem a már meglévő, ismert hatóanyagok származékai. Jelen közleményünk a „Dopaminerg antipszichotikumok kutatása és fejlesztése” című, a Moravcsik Alapítvány által kiadott egyetemi jegyzet „Gyógyszerprofil- összevetés: az in silico farmakológia lehetőségei” című fejezetének rövidített változata.

Zeke András, Garai Ágnes, Reményi Attila

Mitogén-aktivált protein kináz kötő lineáris motívumok azonosítása szerkezeti alapon

BIOKÉMIA 36/2

Az eukarióta mitogén-aktivált protein kináz (MAPK) jelátviteli útvonalak a sejt környezeti ingerekre adott válaszainak talán az egyik legősibb és legszélesebb körben használt szabályozó rendszerei. Cikkünkben bemutatjuk, hogy MAPK-ok partner fehérjékkel alkotott komplexeinek atomi felbontású szerkezetei hogyan járulhatnak hozzá, hogy felderíthessük ennek az ősi jelátviteli rendszernek az organizmus szintű szerveződését, de akár az evolúcióját is.

Garai A, Zeke A, Gógl G, Töro I, Fördos F, Blankenburg H, Bárkai T, Varga J, Alexa A, Emig D, Albrecht M, Reményi A

Specificity of linear motifs that bind to a common mitogen-activated protein kinase docking groove.

IF:7,5

Sci Signal. 2012 Oct 9;5(245):ra74. doi: 10.1126/scisignal.2003004.

Mitogen-activated protein kinases (MAPKs) have a docking groove that interacts with linear "docking" motifs in binding partners. To determine the structural basis of binding specificity between MAPKs and docking motifs, we quantitatively analyzed the ability of 15 docking motifs from diverse MAPK partners to bind to c-Jun amino-terminal kinase 1 (JNK1), p38α, and extracellular signal-regulated kinase 2 (ERK2). Classical docking motifs mediated highly specific binding only to JNK1, and only those motifs with a sequence pattern distinct from the classical MAPK binding docking motif consensus differentiated between the topographically similar docking grooves of ERK and p38α. Crystal structures of four complexes of MAPKs with docking peptides, representing JNK-specific, ERK-specific, or ERK- and p38-selective binding modes, revealed that the regions located between consensus positions in the docking motifs showed conformational diversity. Although the consensus positions in the docking motifs served as anchor points that bound to common MAPK surface features and mostly contributed to docking in a nondiscriminatory fashion, the conformation of the intervening region between the anchor points mostly determined specificity. We designed peptides with tailored MAPK binding profiles by rationally changing the length and amino acid composition of intervening regions located between anchor points. These results suggest a coherent structural model for MAPK docking specificity that reveals how short linear motifs binding to a common kinase docking groove can mediate diverse interaction patterns and contribute to correct MAPK partner selection in signaling networks.

Fodor K, Wolf J, Erdmann R, Schliebs W, Wilmanns M.

Molecular requirements for peroxisomal targeting of alanine-glyoxylate aminotransferase as an essential determinant in primary hyperoxaluria type 1.

IF:11,452

PLoS Biol 10(4):e1001309

Alanine-glyoxylate aminotransferase is a peroxisomal enzyme, of which various missense mutations lead to irreversible kidney damage via primary hyperoxaluria type 1, in part caused by improper peroxisomal targeting. To unravel the molecular mechanism of its recognition by the peroxisomal receptor Pex5p, we have determined the crystal structure of the respective cargo-receptor complex. It shows an extensive protein/protein interface, with contributions from residues of the peroxisomal targeting signal 1 and additional loops of the C-terminal domain of the cargo. Sequence segments that are crucial for receptor recognition and hydrophobic core interactions within alanine-glyoxylate aminotransferase are overlapping, explaining why receptor recognition highly depends on a properly folded protein. We subsequently characterized several enzyme variants in vitro and in vivo and show that even minor protein fold perturbations are sufficient to impair Pex5p receptor recognition. We discuss how the knowledge of the molecular parameters for alanine-glyoxylate aminotransferase required for peroxisomal translocation could become useful for improved hyperoxaluria type 1 treatment.

Simon Z, Peragovics A, Vigh-Smeller M, Csukly G, Tombor L, Yang Z, Zahoránszky-Kohalmi G, Végner L, Jelinek B, Hári P, Hetényi C, Bitter I, Czobor P, Málnási-Csizmadia A.

Drug effect prediction by polypharmacology-based interaction profiling.

IF:3,882

J Chem Inf Model 52:134-45.

Most drugs exert their effects via multitarget interactions, as hypothesized by polypharmacology. While these multitarget interactions are responsible for the clinical effect profiles of drugs, current methods have failed to uncover the complex relationships between them. Here, we introduce an approach which is able to relate complex drug-protein interaction profiles with effect profiles. Structural data and registered effect profiles of all small-molecule drugs were collected, and interactions to a series of nontarget protein binding sites of each drug were calculated. Statistical analyses confirmed a close relationship between the studied 177 major effect categories and interaction profiles of ca. 1200 FDA-approved small-molecule drugs. On the basis of this relationship, the effect profiles of drugs were revealed in their entirety, and hitherto uncovered effects could be predicted in a systematic manner. Our results show that the prediction power is independent of the composition of the protein set used for interaction profile generation.

Gyimesi M, Harami GM, Sarlós K, Hazai E, Bikádi Z, Kovács M.

Complex activities of the human Blooms syndrome helicase are encoded in a core region comprising the RecA and Zn-binding domains.

IF:7,836

Nucleic Acids Res 40:3952-63

Blooms syndrome DNA helicase (BLM), a member of the RecQ family, is a key player in homologous recombination (HR)-based error-free DNA repair processes. During HR, BLM exerts various biochemical activities including single-stranded (ss) DNA translocation, separation and annealing of complementary DNA strands, disruption of complex DNA structures (e.g. displacement loops) and contributes to quality control of HR via clearance of Rad51 nucleoprotein filaments. We performed a quantitative mechanistic analysis of truncated BLM constructs that are shorter than the previously identified minimal functional module. Surprisingly, we found that a BLM construct comprising only the two conserved RecA domains and the Zn(2+)-binding domain (residues 642-1077) can efficiently perform all mentioned HR-related activities. The results demonstrate that the Zn(2+)-binding domain is necessary for functional interaction with DNA. We show that the extensions of this core, including the winged-helix domain and the strand separation hairpin identified therein in other RecQ-family helicases, are not required for mechanochemical activity per se and may instead play modulatory roles and mediate protein-protein interactions.

Peragovics A, Simon Z, Brandhuber I, Jelinek B, Hári P, Hetényi C, Czobor P, Málnási-Csizmadia A.

Contribution of 2D and 3D Structural Features of Drug Molecules in the Prediction of Drug Profile Matching.

IF:3,882

J Chem Inf Model. 2012 Jun 29.

Drug Profile Matching (DPM), a novel virtual affinity fingerprinting method capable of predicting the medical effect profiles of small molecules, was introduced by our group recently. The method exploits the information content of interaction patterns generated by flexible docking to a series of rigidly kept nontarget protein active sites. We presented the ability of DPM to classify molecules excellently, and the question arose, what the contribution of 2D and 3D structural features of the small molecules is to the intriguingly high prediction power of DPM. The present study compared the prediction powers for effect profiles of 1163 FDA-approved drug compounds determined by DPM and ChemAxon 2D and 3D similarity fingerprinting approaches. We found that DPM outperformed the 2D and 3D approaches in almost all therapeutic categories for drug classification except for mechanically rigid structural categories where high accuracy was obtained by all three methods. Moreover, we also tested the predictive power of DPM on external data by reducing the parent data set and demonstrated that DPM can overcome the common screening problems of 2D and 3D similarity methods arising from the presence of structurally diverse molecules in certain effect categories.

Sarlós K, Gyimesi M, Kovács M.

RecQ helicase translocates along single-stranded DNA with a moderate processivity and tight mechanochemical coupling.

IF:9,681

Proc Natl Acad Sci U S A 109:9804-9

Maintenance of genome integrity is the major biological role of RecQ-family helicases via their participation in homologous recombination (HR)-mediated DNA repair processes. RecQ helicases exert their functions by using the free energy of ATP hydrolysis for mechanical movement along DNA tracks (translocation). In addition to the importance of translocation per se in recombination processes, knowledge of its mechanism is necessary for the understanding of more complex translocation-based activities, including nucleoprotein displacement, strand separation (unwinding), and branch migration. Here, we report the key properties of the ssDNA translocation mechanism of Escherichia coli RecQ helicase, the prototype of the RecQ family. We monitored the pre-steady-state kinetics of ATP hydrolysis by RecQ and the dissociation of the enzyme from ssDNA during single-round translocation. We also gained information on the translocation mechanism from the ssDNA length dependence of the steady-state ssDNA-activated ATPase activity. We show that RecQ occludes 18 ± 2 nt on ssDNA during translocation. The hydrolysis of ATP is noncooperative in the presence of ssDNA, indicating that RecQ active sites work independently during translocation. In the applied conditions, the enzyme hydrolyzes 35 ± 4 ATP molecules per second during ssDNA translocation. RecQ translocates at a moderate processivity, with a mean run length of 100-320 nt on ssDNA. The determined tight mechanochemical coupling of 1.1 ± 0.2 ATP consumed per nucleotide traveled indicates an inchworm-type mechanism.

Képiró M, Várkuti BH, Bodor A, Hegyi G, Drahos L, Kovács M, Málnási-Csizmadia A.

Azidoblebbistatin, a photoreactive myosin inhibitor.

IF:9,681

Proc Natl Acad Sci U S A 109:9402-7

Photoreactive compounds are important tools in life sciences that allow precisely timed covalent crosslinking of ligands and targets. Using a unique technique we have synthesized azidoblebbistatin, which is a derivative of blebbistatin, the most widely used myosin inhibitor. Without UV irradiation azidoblebbistatin exhibits identical inhibitory properties to those of blebbistatin. Using UV irradiation, azidoblebbistatin can be covalently crosslinked to myosin, which greatly enhances its in vitro and in vivo effectiveness. Photo-crosslinking also eliminates limitations associated with the relatively low myosin affinity and water solubility of blebbistatin. The wavelength used for photo-crosslinking is not toxic for cells and tissues, which confers a great advantage in in vivo tests. Because the crosslink results in an irreversible association of the inhibitor to myosin and the irradiation eliminates the residual activity of unbound inhibitor molecules, azidoblebbistatin has a great potential to become a highly effective tool in both structural studies of actomyosin contractility and the investigation of cellular and physiological functions of myosin II. We used azidoblebbistatin to identify previously unknown low-affinity targets of the inhibitor (EC(50) ≥ 50 μM) in Dictyostelium discoideum, while the strongest interactant was found to be myosin II (EC(50) = 5 μM). Our results demonstrate that azidoblebbistatin, and potentially other azidated drugs, can become highly useful tools for the identification of strong- and weak-binding cellular targets and the determination of the apparent binding affinities in in vivo conditions.

Malik ZA, Amir S, Venekei I.

SERINE proteinase like activity in apolipophorin III from the hemolymph of desert locust, Schistocerca gregaria.

IF:1,380 http://onlinelibrary.wiley.com/doi/10.1002/arch.21020/abstract;jsessionid=D7C6BC53793886DE8B5E8656DB93F571.d03t01

Arch Insect Biochem Physiol 80:26-41

Apolipophorin III (apoLp-III) has been known as a lipid transport protein of insects. Recent studies indicated the involvement of apoLp-III in immune reactions and in the control of cell destruction, but no enzymatic activity has so far been detected. In the present study, a protease from the hemolymph of Schistocerca gregaria was purified to homogeneity and its enzymatic activity was examined. Identity as chymotrypsin-like proteinase was established by its high affinity toward bulky aromatic substrates and its catalytic specificity for amide or ester bonds on the synthetic substrates, Suc-Ala-Ala-Pro-Xaa-AMC (where Xaa was Phe, Tyr, Trp, and Lys, and AMC is 7-amino-4-methyl-coumarin) and thiolbenzyl ester substrate Suc-Ala-Ala-Pro-Phe-SBzl. The sensitivity for serine protease and chymotrypsin-specific covalent inhibitors, PMSF, TPCK, and noncovalent inhibitors SGCI, showed that it is a chymotrypsin-like proteinase. It showed its maximum activity at pH 8.0 and 55°C for the hydrolysis of Suc-Ala-Ala-Pro-Tyr-AMC. According to similarities in the amino terminal sequence, molar mass (19 kDa) and retention on reversed-phase analytical high-performance liquid chromatography (HPLC) column, this protein is S. gregaria homologue of Locusta migratoria apoLp-III. Our data suggest that apoLp-III also has an inherent proteolytic activity. Results indicated that S. gregaria apoLp-III is a good catalyst and could be used as a biotechnological tool in food processing and in agricultural biotechnology.

Szenes A, Pál G.

Mapping hidden potential identity elements by computing the average discriminating power of individual tRNA positions.

IF:5,164

DNA Res 19:245-58

The recently published discrete mathematical method, extended consensus partition (ECP), identifies nucleotide types at each position that are strictly absent from a given sequence set, while occur in other sets. These are defined as discriminating elements (DEs). In this study using the ECP approach, we mapped potential hidden identity elements that discriminate the 20 different tRNA identities. We filtered the tDNA data set for the obligatory presence of well-established tRNA features, and then separately for each identity set, the presence of already experimentally identified strictly present identity elements. The analysis was performed on the three kingdoms of life. We determined the number of DE, e.g. the number of sets discriminated by the given position, for each tRNA position of each tRNA identity set. Then, from the positional DE numbers obtained from the 380 pairwise comparisons of the 20 identity sets, we calculated the average excluding value (AEV) for each tRNA position. The AEV provides a measure on the overall discriminating power of each position. Using a statistical analysis, we show that positional AEVs correlate with the number of already identified identity elements. Positions having high AEV but lacking published identity elements predict hitherto undiscovered tRNA identity elements.

Héja D, Harmat V, Fodor K, Wilmanns M, Dobó J, Kékesi KA, Závodszky P, Gál P, Pál G.

Monospecific inhibitors show that both mannan-binding lectin-associated serine protease-1 (MASP-1) and -2 Are essential for lectin pathway activation and reveal structural plasticity of MASP-2.

IF:5,328

J Biol Chem 287:20290-300

The lectin pathway is an antibody-independent activation route of the complement system. It provides immediate defense against pathogens and altered self-cells, but it also causes severe tissue damage after stroke, heart attack, and other ischemia reperfusion injuries. The pathway is triggered by target binding of pattern recognition molecules leading to the activation of zymogen mannan-binding lectin-associated serine proteases (MASPs). MASP-2 is considered as the autonomous pathway-activator, while MASP-1 is considered as an auxiliary component. We evolved a pair of monospecific MASP inhibitors. In accordance with the key role of MASP-2, the MASP-2 inhibitor completely blocks the lectin pathway activation. Importantly, the MASP-1 inhibitor does the same, demonstrating that MASP-1 is not an auxiliary but an essential pathway component. We report the first Michaelis-like complex structures of MASP-1 and MASP-2 formed with substrate-like inhibitors. The 1.28 â„« resolution MASP-2 structure reveals significant plasticity of the protease, suggesting that either an induced fit or a conformational selection mechanism should contribute to the extreme specificity of the enzyme.

Héja D, Kocsis A, Dobó J, Szilágyi K, Szász R, Závodszky P, Pál G, Gál P.

Revised mechanism of complement lectin-pathway activation revealing the role of serine protease MASP-1 as the exclusive activator of MASP-2.

IF:9,681

Proc Natl Acad Sci U S A 109:10498-503

The lectin pathway of complement activation is an important component of the innate immune defense. The initiation complexes of the lectin pathway consist of a recognition molecule and associated serine proteases. Until now the autoactivating mannose-binding lectin-associated serine protease (MASP)-2 has been considered the autonomous initiator of the proteolytic cascade. The role of the much more abundant MASP-1 protease was controversial. Using unique, monospecific inhibitors against MASP-1 and MASP-2, we corrected the mechanism of lectin-pathway activation. In normal human serum, MASP-2 activation strictly depends on MASP-1. MASP-1 activates MASP-2 and, moreover, inhibition of MASP-1 prevents autoactivation of MASP-2. Furthermore we demonstrated that MASP-1 produces 60% of C2a responsible for C3 convertase formation.

Kapp GT, Liu S, Stein A, Wong DT, Reményi A, Yeh BJ, Fraser JS, Taunton J, Lim WA, Kortemme T.

Control of protein signaling using a computationally designed GTPase/GEF orthogonal pair.

IF:9,681

Proc Natl Acad Sci U S A 109:5277-82.

Signaling pathways depend on regulatory protein-protein interactions; controlling these interactions in cells has important applications for reengineering biological functions. As many regulatory proteins are modular, considerable progress in engineering signaling circuits has been made by recombining commonly occurring domains. Our ability to predictably engineer cellular functions, however, is constrained by complex crosstalk observed in naturally occurring domains. Here we demonstrate a strategy for improving and simplifying protein network engineering: using computational design to create orthogonal (non-crossreacting) protein-protein interfaces. We validated the design of the interface between a key signaling protein, the GTPase Cdc42, and its activator, Intersectin, biochemically and by solving the crystal structure of the engineered complex. The designed GTPase (orthoCdc42) is activated exclusively by its engineered cognate partner (orthoIntersectin), but maintains the ability to interface with other GTPase signaling circuit components in vitro. In mammalian cells, orthoCdc42 activity can be regulated by orthoIntersectin, but not wild-type Intersectin, showing that the designed interaction can trigger complex processes. Computational design of protein interfaces thus promises to provide specific components that facilitate the predictable engineering of cellular functions.

Wojtasz L, Cloutier JM, Baumann M, Daniel K, Varga J, Fu J, Anastassiadis K, Stewart AF, Reményi A, Turner JM, Tóth A.

Meiotic DNA double-strand breaks and chromosome asynapsis in mice are monitored by distinct HORMAD2-independent and -dependent mechanisms.

IF:13,892

Genes Dev 26:958-73

Meiotic crossover formation involves the repair of programmed DNA double-strand breaks (DSBs) and synaptonemal complex (SC) formation. Completion of these processes must precede the meiotic divisions in order to avoid chromosome abnormalities in gametes. Enduring key questions in meiosis have been how meiotic progression and crossover formation are coordinated, whether inappropriate asynapsis is monitored, and whether asynapsis elicits prophase arrest via mechanisms that are distinct from the surveillance of unrepaired DNA DSBs. We disrupted the meiosis-specific mouse HORMAD2 (Hop1, Rev7, and Mad2 domain 2) protein, which preferentially associates with unsynapsed chromosome axes. We show that HORMAD2 is required for the accumulation of the checkpoint kinase ATR along unsynapsed axes, but not at DNA DSBs or on DNA DSB-associated chromatin loops. Consistent with the hypothesis that ATR activity on chromatin plays important roles in the quality control of meiotic prophase, HORMAD2 is required for the elimination of the asynaptic Spo11(-/-), but not the asynaptic and DSB repair-defective Dmc1(-/-) oocytes. Our observations strongly suggest that HORMAD2-dependent recruitment of ATR to unsynapsed chromosome axes constitutes a mechanism for the surveillance of asynapsis. Thus, we provide convincing evidence for the existence of a distinct asynapsis surveillance mechanism that safeguards the ploidy of the mammalian germline.

Pálfy M, Reményi A, Korcsmáros T.

Endosomal crosstalk: meeting points for signaling pathways.

IF:12,354

Trends Cell Biol. 2012 Jul 13

Gáspári, Z., Süveges, D., Perczel, A., Nyitray, L., and Tóth, G.

Charged single alpha-helices in proteomes revealed by a consensus prediction approach.

IF:3,635

Biochem Biophys Acta Proteins and Proteomics 1824:637-646

Charged single α-helices (CSAHs) constitute a recently recognized protein structural motif. Its presence and role is characterized in only a few proteins. To explore its general features, a comprehensive study is necessary. We have set up a consensus prediction method available as a web service (at http://csahserver.chem.elte.hu) and downloadable scripts capable of predicting CSAHs from protein sequences. Using our method, we have performed a comprehensive search on the UniProt database. We found that the motif is very rare but seems abundant in proteins involved in symbiosis and RNA binding/processing. Although there are related proteins with CSAH segments, the motif shows no deep conservation in protein families. We conclude that CSAH-containing proteins, although rare, are involved in many key biological processes. Their conservation pattern and prevalence in symbiosis-associated proteins suggest that they might be subjects of relatively rapid molecular evolution and thus can contribute to the emergence of novel functions.

Bence Kiss, Annette Duelli, László Radnai, Katalin A. Kékesi, Gergely Katona, László Nyitray

Crystal structure of the S100A4-myosin IIA tail fragment complex reveals an asymmetric target binding mechanism

Proc. Natl. Acad. Sci. USA, 109(16):6048-53

S100A4 is a member of the S100 family of calcium-binding proteins that is directly involved in tumor metastasis. It binds to the nonmuscle myosin IIA (NMIIA) tail near the assembly competence domain (ACD) promoting filament disassembly, which could be associated with increasing metastatic potential of tumor cells. Here, we investigate the mechanism of S100A4-NMIIA interaction based on binding studies and the crystal structure of S100A4 in complex with a 45-residue-long myosin heavy chain fragment. Interestingly, we also find that S100A4 binds as strongly to a homologous heavy chain fragment of nonmuscle myosin IIC as to NMIIA. The structure of the S100A4-NMIIA complex reveals a unique mode of interaction in the S100 family: A single, predominantly α-helical myosin chain is wrapped around the Ca(2+)-bound S100A4 dimer occupying both hydrophobic binding pockets. Thermal denaturation experiments of coiled-coil forming NMIIA fragments indicate that the coiled-coil partially unwinds upon S100A4 binding. Based on these results, we propose a model for NMIIA filament disassembly: Part of the random coil tailpiece and the C-terminal residues of the coiled-coil are wrapped around an S100A4 dimer disrupting the ACD and resulting in filament dissociation. The description of the complex will facilitate the design of specific drugs that interfere with the S100A4-NMIIA interaction.

Várkuti BH, Yang Z, Kintses B, Erdélyi P, Bárdos-Nagy I, Kovács AL, Hári P, Kellermayer M, Vellai T, Málnási-Csizmadia A.

A novel actin binding site of myosin required for effective muscle contraction.

Nat. Struct. Mol. Biol. 2012 Feb 12;19(3):299-306.