Structure-Function Relations in Oxaloacetate Decarboxylase Complex. Fluorescence and Infrared Approaches to Monitor Oxomalonate and Na+ Binding Effect

ions across the membrane, which drives endergonic membrane reactions such as ATP synthesis, transport and motility. OAD is a membrane-bound enzyme composed of α, β and γ subunits. The α subunit contains the carboxyltransferase catalytic site. characteristic of a high content of α helix structures. Addition of oxomalonate induced a shift of the amide-I band of OAD toward higher wavenumbers, interpreted as a slight decrease of β sheet structures and a concomitant increase of α helix structures. Oxomalonate binding to αγand α subunits also provoked secondary structure variations, but these effects were negligible compared to OAD complex. alters the tryptophan environment of the β subunit, consistent with the function of these subunits within the enzyme complex. Formation of a complex between OAD and its substrates elicits structural changes in the α-helical as well as β-strand secondary structure elements

Relation structure-fonction dans la famille des guanidino kinases

La première partie de cette thèse décrit la dénaturation comparée de plusieurs guanidino kinases (GdnK) de structures quaternaires variées (monomère, dimère ou octamère). Ces enzymes catalysent le transfert réversible du phosphate g de l ATP vers un accepteur guanidylique (créatine, arginine). Nous avons montré que, pour les créatine kinases multimériques, l association des sous-unités est requise pour l expression de l activité enzymatique mais que cette association ne leur apporte pas une plus grande stabilité conformationnelle par rapport à une arginine kinase monomérique. Dans la seconde partie, nous décrivons la caractérisation d une nouvelle guanidino kinase à la structure dupliquée qui est exprimée dans les cercaires de Schistosoma mansoni. Nous avons mis au point un protocole d expression et de purification pour cette enzyme, identifié ses substrats physiologiques, défini les paramètres enzymatiques pour ces substrats dans le sens de formation du phosphagène, cristallisé cette enzyme et résolu sa structure tridimensionnelle par diffraction des rayons XThe first part of this thesis describes the comparative unfolding of guanidino kinases with differing quaternary structure (monomer, dimer or octamer). These enzymes catalyze the reversible transfer of the ATP g phosphate to a guanidylic acceptor (creatine, arginine). We have shown, that for dimer or octamer creatine kinases, association of subunits is required for expression of catalytic activity. However, a higher level of quaternary structure does not provide these enzymes an increased conformational stability as compared to a monomeric arginine kinase. In a second part, we describe the characterization of a new guanidino kinase displaying a duplicated structure and which is expressed in Schistosoma mansoni cercariae. We have developed expression and purification protocols for the untagged protein, identified its physiological substrates, defined its enzymatic parameters in the direction of phosphagene formation, crystallized this enzyme and determined its three-dimensional structure by X-ray diffractionLYON1-BU.Sciences (692662101) / SudocSudocFranceF

La créatine kinase mitochondriale - organisatrice de la membrane mitochondriale ? (ségrégation de la cardiolipine sur des monocouches phospholipidiques)

En utilisant comme modèle membranaire les monocouches de Langmuir à l interface air-tampon et des techniques de caractérisation spécifiques, nous avons obtenu des nouvelles informations sur l interaction entre la créatine kinase mitochondriale et la membrane interne de la mitochondrie. Par microscopie à l angle de Brewster nous avons pu visualiser la formation de complexes spécifiques cardiolipine-CKmt, avec ségrégation de la cardiolipine dans la membrane biomimétique. La fixation de la CKmt a lieu avec insertion de domaines protéiques parmi les lipides et abouti à une stabilisation globale du film interfacial, comme indiqué par des mesures de capacité différentielle d une électrode de mercure. Une réorientation des domaines protéiques, principalement des hélices a, a également pu être mise en évidence par spectroscopie infrarouge de réflexion-absorption par modulation de la polarisation (PM-IRRAS). L effet de la CKmt sur la morphologie de la monocouche dépend du degré d insaturation des chaînes acyles. Ceci est en relation avec la forte proportion d acide linoléique (C18:2), caractéristique des molécules de cardiolipine dans les cellules musculaires. Le phénomène de ségrégation induit par la CKmt, pourrait contribuer à la modulation de la distribution de cardiolipine et avoir des conséquences sur la fixation d autres molécules comme le cytochrome c ou les protéines de la famille Bcl-2. La membrane mitochondriale étant siège des nombreux phénomènes de peroxydation, de part sa structure et sa localisation, la CKmt est une cible des molécules oxydantes générées. Parmi ces molécules, le 4-hydroxynonénal, inactive l enzyme, modifie sa structure et induit l accumulation de complexes protéiques agrégés au niveau de la membrane. L interaction de la CKmt avec la membrane biomimétique est fortement perturbée, aussi bien en monocouche que sur les liposomesaThe interaction between the mitochondrial isoform of creatine kinase (mtCK) and a mitochondrial inner membrane biomimetic model was analysed using phospholipid monolayers at the air-buffer interface. New insight on the structural and morphological consequences both on protein and membrane was obtained. As visualised by Brewster angle microscopy, mtCK induces formation of specific protein cardiolipin complexes, leading to the segregation of cardiolipin molecules. MtCK binding occurs with partial insertion between lipids and results in an overall stabilisation of the membrane, as indicated by differential capacity measurements. A reorientation of protein -helical structures was also observed by polarisation modulation infrared reflexion-absorption spectroscopy (PM-IRRAS). Formation of mtCK cardiolipin complexes was found to be highly dependable of acyl chain unsaturation. This is of physiological relevance as one of the main characteristics of cardiolipin in muscle mitochondria is the high percentage of linoleic acid (C18:2). MtCK-induced segregation may contribute to membrane organisation by modulating cardiolipin distribution, and consequently membrane binding of other molecules such as cytochrome c or Bcl-2 family proteins. Mitochondrial membranes are the site of numerous peroxydation processes. MtCK structure and localisation in the vicinity of the membrane make it a target for oxidative molecules produced during such processes. Among these molecules, 4-hydroxynonenal, induces enzyme inactivation, modifies its structure and induces accumulation of aggregates at membranes. MtCK binding to model membranes, liposomes or monolayers, is thus disturbed. All these complex perturbation were analysed and debated under a physiological point of view along this thesis workLYON1-BU.Sciences (692662101) / SudocSudocFranceF

Structural comparison of highly similar nucleoside diphosphate kinases: molecular explanation of distinct membrane binding behavior

International audienceNDPK-A, NDPK-B and NDPK-D are three enzymes which belong to the NDPK group I isoforms and are not only involved in metabolism process but also in transcriptional regulation, DNA cleavage, histidine protein kinase activity and metastasis development. Those enzymes were reported to bind to membranes either in mitochondria where NDPK-D influences cardiolipin lateral organization and is thought to be involved in apoptotic pathway or in cytosol where NDPK-A and NDPK-B membrane association was shown to influence several cellular processes like endocytosis, cellular adhesion, ion transport, etc. However, despite numerous studies, the role of NDPK-membrane association and the molecular details of the binding process are still elusive. In the present work, a comparative study of the three NDPK isoforms allowed us to show that although membrane binding is a common feature of these enzymes, mechanisms differ at the molecular scale. NDPK-A was not able to bind to model membranes mimicking the inner leaflet of plasma membrane, suggesting that its in vivo membrane association is mediated by a non-lipidic partner or other partners than the studied phospholipids. On the contrary, NDPK-B and NDPK-D were shown to bind efficiently to liposomes mimicking plasma membrane and mitochondrial inner membrane respectively but details of the binding mechanism differ between the two enzymes as NDPK-B binding necessarily involved an anionic phospholipid partner while NDPK-D can bind either zwitterionic or anionic phospholipids. Although sharing similar secondary structure and homohexameric quaternary arrangement, tryptophan fluorescence revealed fine disparities in NDPK tertiary structures. Interfacial behavior as well as ANS fluorescence showed further dissimilarities between NDPK isoforms, notably the presence of distinct accessible hydrophobic areas as well as different capacity to form Gibbs monolayers related to their surface activity properties. Those distinct features may contribute to explain the differences in the protein behavior towards membrane binding

Hydrogen/deuterium exchange studies of native rabbit MM-CK dynamics

Creatine kinase (CK) isoenzymes catalyse the reversible transfer of a phosphoryl group from ATP onto creatine. This reaction plays a very important role in the regulation of intracellular ATP concentrations in excitable tissues. CK isoenzymes are highly resistant to proteases in native conditions. To appreciate localized backbone dynamics, kinetics of amide hydrogen exchange with deuterium was measured by pulse-labeling the dimeric cytosolic muscle CK isoenzyme. Upon exchange, the protein was digested with pepsin, and the deuterium content of the resulting peptides was determined by liquid chromatography coupled to mass spectrometry (MS). The deuteration kinetics of 47 peptides identified by MS/MS and covering 96% of the CK backbone were analyzed. Four deuteration patterns have been recognized: The less deuterated peptides are located in the saddle-shaped core of CK, whereas most of the highly deuterated peptides are close to the surface and located around the entrance to the active site. Their exchange kinetics are discussed by comparison with the known secondary and tertiary structures of CK with the goal to reveal the conformational dynamics of the protein. Some of the observed dynamic motions may be linked to the conformational changes associated with substrate binding and catalytic mechanism

Dynamical properties of the loop 320s of substrate-free and substrate-bound muscle creatine kinase by NMR

International audienceMuscle creatine kinase (MCK; ) is a 86 kDa homodimer that belongs to the family of guanidino kinases. MCK has been intensively studied for several decades, but it is still not known why it is a dimer because this quaternary structure does not translate into obvious structural or functional advantages over the homologous monomeric arginine kinase. In particular, it remains to be demonstrated whether MCK subunits are independent. Here, we describe NMR chemical-shift perturbation and relaxation experiments designed to study the active site 320s flexible loop of this enzyme. The analysis was performed with the enzyme in its ligand-free and MgADP-complexed forms, as well as with the transition-state analogue abortive complex (MCKMgADPcreatinenitrate ion). Our data indicate that each subunit can bind substrates independently

Etude structurale et dynamique de la créatine kinase (CK-MM) par RMN

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Magnesium-Adenosine Diphosphate binding sites in wild-type creatine kinase and in mutants:role of aromatic residues probed by Raman and Infrared spectroscopies

Two distinct methods were used to investigate the role of Trp residues during Mg-ADP binding to cytosolic creatine kinase (CK) from rabbit muscle: (1) Raman spectroscopy, which is very sensitive to the environment of aromatic side-chain residues, and (2) reaction-induced infrared difference spectroscopy (RIDS) and photolabile substrate (ADP[Et(PhNO2)]), combined with site-directed mutagenesis on the four Trp residues of CK. Our Raman results indicated that the environment of Trp and of Tyr were not affected during Mg-ADP binding to CK. Analysis of RIDS of wild-type CK, inactive W227Y, and active W210,217,272Y mutants suggested that Trp227 was not involved in the stacking interactions. Results are consistent with Trp227 being essential to prevent water molecules from entering in the active site [as suggested by Gross, M., Furter-Graves, E. M., Wallimann, T., Eppenberger, H. M., and Furter, R. (1994) Protein Sci. 3, 1058−1068] and that another Trp could in addition help to steer the nucleotide in the binding site, although it is not essential for the activity of CK. Raman and infrared spectra indicated that Mg-ADP binding does not involve large secondary structure changes. Only 3−4 residues absorbing in the amide I region are directly implicated in the Mg-ADP binding (corresponding to secondary structure changes less than 1%), suggesting that movement of protein domains due to Mg-nucleotide binding do not promote large secondary structure changes