Localization and characterization of the inhibitory Ca2+-binding site of Physarum polycephalum myosin II.

A myosin II is thought to be the driving force of the fast cytoplasmic streaming in the plasmodium of Physarum polycephalum. This regulated myosin, unique among conventional myosins, is inhibited by direct Ca2+ binding. Here we report that Ca2+ binds to the first EF-hand of the essential light chain (ELC) subunit of Physarum myosin. Flow dialysis experiments of wild-type and mutant light chains and the regulatory domain revealed a single binding site that shows moderate specificity for Ca2+. The regulatory light chain, in contrast to regulatory light chains of higher eukaryotes, is unable to bind divalent cations. Although the Ca2+-binding loop of ELC has a canonical sequence, replacement of glutamic acid to alanine in the -z coordinating position only slightly decreased the Ca2+ affinity of the site, suggesting that the Ca2+ coordination is different from classical EF-hands; namely, the specific "closed-to-open" conformational transition does not occur in the ELC in response to Ca2+. Ca2+- and Mg2+-dependent conformational changes in the microenvironment of the binding site were detected by fluorescence experiments. Transient kinetic experiments showed that the displacement of Mg2+ by Ca2+ is faster than the change in direction of cytoplasmic streaming; therefore, we conclude that Ca2+ inhibition could operate in physiological conditions. By comparing the Physarum Ca2+ site with the well studied Ca2+ switch of scallop myosin, we surmise that despite the opposite effect of Ca2+ binding on the motor activity, the two conventional myosins could have a common structural basis for Ca2+ regulation

Myosin V from Drosophila reveals diversity of motor mechanisms within the myosin V family

Myosin V is the best characterized vesicle transporter in vertebrates, but it has been unknown as to whether all members of the myosin V family share a common, evolutionarily conserved mechanism of action. Here we show that myosin V from Drosophila has a strikingly different motor mechanism from that of vertebrate myosin Va, and it is a nonprocessive, ensemble motor. Our steady-state and transient kinetic measurements on single-headed constructs reveal that a single Drosophila myosin V molecule spends most of its mechanochemical cycle time detached from actin, therefore it has to function in processive units that comprise several molecules. Accordingly, in in vitro motility assays, double-headed Drosophila myosin V requires high surface concentrations to exhibit a continuous translocation of actin filaments. Our comparison between vertebrate and fly myosin V demonstrates that the well preserved function of myosin V motors in cytoplasmic transport can be accomplished by markedly different underlying mechanisms

Miozin motorfehérjék: szerkezet, funkció, szabályozás, kötőpartnerek = Myosin motor proteins: structure, function, regulation, binding partners

A pályázat során a miozin motorfehérjék szerkezet-funkció összefüggéseit vizsgáltuk. A pályázat talán legfontosabb eredménye, hogy elsőnek sikerült atomi felbontású képet kapnunk a miozin coiled-coil (alfa-helikális dimer) szerkezetű farok régió egy részletéről, ráadásul egy olyan instabil doménről, amely a konvencionális miozin (II-es típus) működésében és szabályozásában is fontos szerepet játszhat. A szabályozás kapcsán továbbá meghatároztuk egy Ca2+-ionokkal gátolt miozin II regulációs doménjánek térszerkezetét. Egy, a miozin motor doménben található, régóta ismert reaktív lizin oldalláncról bizonyítottuk, hogy konformációs szenzorként alkalmazható az összes miozin II-ben. Érdekes új eredménynek tekintjük, hogy a VI-os típusú miozin farok régiójában azonosítottunk egy olyan, erősen töltött molekula-részletet, amely egyszálú stabil alfa-hélixet alkot. Ezt a szerkezeti motívumot in silico módszerekkel sok más fehérjében is megtaláltuk. Az V-ös típusú, intracelluláris transzporter-ként működő miozin Drosophila ortológjának ATPáz kinatikáját vizsgálva azt találtuk, hogy a gerinces miozin V-től eltérően ez a motorfehérje nem processzív működésű. A gerinces miozin V-nél a teher kötésében résztvevő adapter molekula, az ún. dinein könnyű lánc kötőhelyét azonosítottuk, és a dimer farok régióra gyakorolt stablizáló hatását mutattuk ki. | In this project we have conducted structure-function studies on myosin motor proteins. Perhaps the most important result is that we have obtained the first atomic resolution picture of the coiled-coil (alpha-helical dimer) myosin tail, and what is more the structure of an unstable domain having important role in the function and regulation of conventional myosin (type II). Regarding regulation, we have determined the 3-dimensional structure of a regulatory domain from a Ca2+-inhibited myosin II. We have proved that a long-time known reactive lysine of the motor domain can be used as a conformational sensor in all of myosin IIs. An interesting new result is that we have identified a highly charged segment in the tail domain of myosin VI that forms a stable single stranded alpha-helix. This structural motif has been found in several other proteins by in silico methods. Studying the ATPase kinetics of Drosophila myosin V, we have shown that this motor is not processive unlike its vertebrate orthologs. We have localized the binding site of the dynein light chain, a cargo binding adapter subunit, on vertebrtae myosin V, and have shown that it stabilizes the structure of the dimeric tail domain

Real-time kinetic method to monitor isopeptidase activity of transglutaminase 2 on protein substrate

Transglutaminase 2 (TG2) is a ubiquitously expressed multifunctional protein with Ca2+-dependent transamidase activity forming protease resistant Nε-(γ-glutamyl)lysine crosslinks between proteins. It can also function as an isopeptidase cleaving the previously formed crosslinks. The biological significance of this activity has not been revealed yet mainly because of the lack of protein based method for its characterization. Here we report development of a novel kinetic method for measuring isopeptidase activity of human TG2 by monitoring decrease in the fluorescence polarisation of a protein substrate previously formed by crosslinking fluorescently labelled glutamine donor FLpepT26 to S100A4 at a specific lysine residue. The developed method could be applied to test mutant enzymes and compounds which influence isopeptidase activity of TG2

Selective 1Hα NMR methods to reveal functionally relevant proline cis/trans isomers in IDPs

It is important to identify proline cis/trans isomers that appear in several regulatory mechanisms of proteins, and to characterize minor species that are present due to the conformational heterogeneity in intrinsically disordered proteins (IDPs). To obtain residue level information on these mobile systems we introduce two H-1(alpha)-detected, proline selective, real-time homodecoupled NMR experiments and analyze the proline abundant transactivation domain of p53. The measurements are sensitive enough to identify minor conformers present in 4-15 % amounts; moreover, we show the consequences of CK2 phosphorylation on the cis/trans-proline equilibrium. Using our results and available literature data we perform a statistical analysis on how the amino acid type effects the cis/trans-proline distribution. The methods are applicable under physiological conditions, they can contribute to find key proline isomers in proteins, and statistical analysis results may help in amino acid sequence optimization for biotechnological purposes

Jelátviteli kompartmentek és proteolózis az immunválasz, valamint az immun- és idegrendszer közötti kommunikáció szabályozásában = Signaling compartments and proteolysis in regulation of immune responses and the communication between the immune- and nervous systems

Több új immunreceptor-kölcsönhatást (FcR/CR/BCR; sejthalál-R/TCR), és több szabályozó mechanizmust azonosítottunk a lipid raftok és más membrán mikro-kompartmentek, valamint limfocita adaptor fehérjék részéről, melyek fontosak a limfociták effektor funkcióiban ill. a sejthalál folyamatában. Leírtuk az utóbbi folyamatokban kritikus miozin motorfehérje izotípusok néhány molekuláris kapcsolatának szerkezeti hátterét és szabályozási lehetőségét. Kimutattuk egyes komplement (C) faktorok és receptorok sokrétű szerepét az immunválasz szabályozásában és, hogy a belőlük származtatott peptidekkel az allergiás hízósejtválasz gátolható. Leírtuk a C-rendszer szabályozó szerepét a Sclerosis Multiplex (SM) állatmodelljében is. Kimutattuk a tripszin-szerű proteáz aktivitás szabályozó szerepét a B-sejt válaszban, és valószínűsítettük szerepüket a mielin bázikus fehérje specifikus hasításában, mely fontos eleme lehet az SM kialakulásának. Eredményeink alapját képezik szelektív immunmodulánsok kifejlesztésének autoimmun ill. allergiás kórképekben. Kimutattunk egyes immunrendszeri effektor- és idegrendszeri funkciók között fellépő, a citokin hálózat ill. steroid hormonok (ösztrogének, glukortikoidok) útján megvalósuló kommunikációs útvonalakat. Új módszereket is kifejlesztettünk (pl. antigén-targetingre alkalmas egyláncú ellenanyag konstrukciók) és a projekt támogatásával beszereztünk egy konfokális mikroszkópot, melyen több új metodikát optimalizáltunk a celluláris kommunikáció vizsgálatára. | We identified several novel immunoreceptor cross-talk elements (FcR/CR/BCR; cell death-R/TCR) and regulatory mechanisms by lipid rafts, other membrane-compartments, as well as adaptor proteins, which are essential in the lymphocytes' effector functions and cell death. Structural and regulatory aspects of some important molecular interactions of myosin motor protein isotypes, essential players in the above processes, were also described. Multiple regulatory functions of the complement system in the immune response and the inhibitory potential of C3a-derived peptides in the allergic mast cell response were identified. The regulatory potential of the complement system was also shown in an animal model of Sclerosis Multiplex (SM). We have shown that trypsin-like protease activities are involved the B cell response through formation of soluble receptors and that trypsin-4, specifically cleaving MBP, may be a critical element in development of SM. Our results form a basis for development of selective immunomodulators for autoimmune- and allergic diseases. We explored several novel communication pathways between effector mechanisms of the immune system and certain nervous system functions, through the inflammatory cytokine network and steroid hormones. New methods and molecular constructs (e.g. antigen-targeting by engineered single-chain antibodies) and several modern confocal imaging techniques for studying cellular communication were also developed with the support of the grant

Ezrin interacts with S100A4 via both its N- and C-terminal domains

Ezrin belongs to the ERM (ezrin, radixin, moesin) protein family that has a role in cell morphology changes, adhesion and migration as an organizer of the cortical cytoskeleton by linking actin filaments to the apical membrane of epithelial cells. It is highly expressed in a variety of human cancers and promotes metastasis. Members of the Ca2+-binding EF-hand containing S100 proteins have similar pathological properties; they are overexpressed in cancer cells and involved in metastatic processes. In this study, using tryptophan fluorescence and stopped-flow kinetics, we show that S100A4 binds to the N-terminal ERM domain (N-ERMAD) of ezrin with a micromolar affinity. The binding involves the F2 lobe of the N-ERMAD and follows an induced fit kinetic mechanism. Interestingly, S100A4 binds also to the unstructured C-terminal actin binding domain (C-ERMAD) with similar affinity. Using NMR spectroscopy, we characterized the complex of S100A4 with the C-ERMAD and demonstrate that no ternary complex is simultaneously formed with the two ezrin domains. Furthermore, we show that S100A4 co-localizes with ezrin in HEK-293T cells. However, S100A4 very weakly binds to full-length ezrin in vitro indicating that the interaction of S100A4 with ezrin requires other regulatory events such as protein phosphorylation and/or membrane binding, shifting the conformational equilibrium of ezrin towards the open state. As both proteins play an important role in promoting metastasis, the characterization of their interaction could shed more light on the molecular events contributing to this pathological process