Blanc SIMI 7 - Sciences de l'information, de la matière et de l'ingénierie : Chimie moléculaire, organique, de coordination, catalyse et chimie biologique

Etudes structurales d’une interaction fortement spécifique du type lipide-protéine membranaire : implications dans la régulation de la dynamique membranaire – ProLipIn

Etudes structurales d’une interaction fortement spécifique du type lipide-protéine membranaire :

implications dans la régulation de la dynamique membranaire ?

Structural basis for the highly specific interactions between a transmembrane protein and sphingomyelin.

The sorting of proteins and lipids between different organelles is a key requirement for the proper functioning of cells. Membrane traffic involves the shuttling of specialized (COPI) vesicles between the ER and the Golgi and a number of proteins, including p24, a type I membrane protein, have been described that are involved in regulating this function. Recently, a highly specific membrane protein-lipid interactions between p24 and SM-C18 that is involved in lipid sorting and protein oligomerisation, has been identified. The interaction is unique in that the lipid head group as well as the length of the fatty acyl chain have a pronounced influence on the affinity between the lipid and the protein and this high specificity is suggestive for a regulatory role comparable to protein-protein or protein-nucleic acid interactions. We propose to determine the structure of the lipid interaction site of a type-I membrane protein p24 in its bilayer environment in the absence and in the presence of its highly specific lipidic ligand. We aim at defining in detail the interaction sites between the protein and the lipid and thereby reveal the molecular basis for its high specificity and strength. As this is the first protein-lipid interaction known to date with high specificity for both the head group and the fatty chain of the lipid the structural data promise to reveal new fundamental knowledge concerning the interactions and regulation of membrane proteins and a potential novel regulatory role of lipids in bilayer environments.

We propose to determine the structure of the lipid interaction site of a type-I membrane protein p24 in its bilayer environment in the absence and in the presence of its highly specific lipidic ligand using biochemical, optical, multidimensional solution and two different solid-state NMR spectroscopic approaches. In order to investigate the detailed structure of p24 and of its complex with SM C18 we will use three different NMR approaches, each with different requirements for sample preparation and which deliver partly overlapping but also highly complementary information. This will assure backup and control, but even more important provide a more extensive and complete data set for structural analysis than could be obtained from one method alone. The project involves a close interaction between the German and the French partner laboratories with clearly defined tasks. The protein will be prepared and labelled with stable isotopes and reconstituted into lipid bilayers of variable composition where the previous experience of both laboratories will enter to advance the work in the best possible manner. Oriented solid-state NMR spectroscopy offers the tools for an independent characterization of the protein structure and which provides information on the tilt and rotational pitch angles within oriented bilayers, as well as structural and topological alterations due to the presence of specific lipid interactions. These experiments are complemented by MAS solid-state and solution-state NMR experiments of p24 in bilayers in order to characterize in detail the structure of the protein, as well as conformational changes in the presence of SM C18.

We have started to characterize the specific interactions between the transmembrane domain of the p24 protein and the lipid sphingomyelin 18 :0. A peptide of 31 residues additional designed, which contains the p24 transmembrane domain (TMD) of 21 amino acids, some few amino acids on both sides and two lysines at the very N-terminus to improve solubility. A protocol for the preparation of this sequence by solid phase synthesis (SPS) and HPLC was established. Several sequences could be prepared also including the labeling at a number of positions with 2H, 15N.
In a parallel step the conditions for p24TMD reconstitution in lipids bilayer were established.
With these tools developed we were in a position to investigate p24TMD in uniaxially oriented membranes of POPC, POPC/SM (95/5) or POPC/SM (90/10) by 15N or 2H solid state NMR spectroscopy. The 15N spectra obtained in POPC and POPC/SM (95/5) indicate that p24TMD adopts a well defined transmembrane orientation. Notably the alignment of the peptide backbone is not affected by the presence of SM. The 2H spectral lineshape are quite sensitive to peptide dynamics and indicative of a large conformational/orientational distribution at the level of the labeled alanine, a property that is reduced when p24 interacts with SM C18:0. The chemical shift orientational restrictions obtained from each single isotope label at different positions, were used to determine the pitch and tilt angles of the helix accurately.

To have more structural information, we need the fully labelled peptide. This would be unrealistic to achieve by SPPS and we started to work on the cloning and bacterial over-expression in order to establish an alternative method to conveniently produce selectively and u uniformly labeled polypeptides. We tested two bacterial over-expression systems:
- One developed in our laboratory for toxic proteins, in particular antimicrobial peptides. This system uses the pTIXP4 plasmid which carry the TAF12 tag for neutralizing the protein in inclusion bodies. The p24TMD gene including restriction sites was amplified using a dedicated vector, cut and inserted in an expression vector (pTIXP4). The expression was tested in rich medium and the fusion protein found in the membrane fractions.
- A recombinant pMal vector which expresses the MBP-p24TMDH fusion protein was received from our collaborators in Heidelberg. Expression of the fusion protein in rich medium was fine and we thereafter tested the system in minimal medium for isotopic labeling. Protein expression was tested at different temperature and time, and the results indicate that protein induction is most efficient at 18°C and during 24h. After the separation of different fractions, the fusion protein was purified by amylose affinity chromatography. A Tev cleavage site was introduced to cleave the MBP and obtain the p24TMH. We are currently testing different conditions for cleavage.
Having this material at hand will allow us to pursue NMR investigations revealing improved structural details

1. Patricia Kemayo, Christopher Aisenbrey, Evgeniy Salnikov, Britta Brügger, Burkhard Bechinger ‘NMR studies of highly specific lipid-protein interactions in lipid bilayers’ poster, Réunion plénière, GDR 3334 SupraMemBio : Assemblages supramoléculaires et membranes biologiques: Concepts, modèles et fonctions, Fournols, 14-17 October 2012

2. Patricia Kemayo, Christopher Aisenbrey, Evgeniy Salnikov, Britta Brügger, Burkhard Bechinger ‘NMR studies of highly specific lipid-protein interactions in lipid bilayers’ poster, Conference of the société française de biophysique (SFB) and the société française de biochimie et de biologie moléculaire (SFBBM), Grenoble, 21-23 November 2012 (winner of poster prize)

3. Kemayo, P., Aisenbrey, C., Glattard, E., Salnikov, E., and Bechinger, B. «Structural characterisation of highly specific membrane protein-lipid interactions involved in trafficking. A novel regulatory mechanism in membrane dynamics?«
presentation, Workshop of the German-French PhD College
“Membranes and Membrane Proteins” UFA 0407. Strasbourg, France, 30 November 2012.

4. Kemayo, P., Aisenbrey, C., Glattard, E., Salnikov, E., and Bechinger, B. «Structural characterisation of highly specific membrane protein-lipid interactions involved in trafficking. A novel regulatory mechanism in membrane dynamics?«
presentation, ANR awardee mid-term workshop of the projects selected in 2010 in the calls Blanc and Jeunes chercheurs, by the committees SIMI7 (chimie moléculaire, organique, de coordination, catalyse et chimie biologique) et SIMI8 (chimie du solide, colloïdes, physico-chimie). Strasbourg, France, 7 December 2012

La répartition des protéines et lipides entre les différentes organelles est un élément clé pour le bon fonctionnement cellulaire. Le trafic membranaire implique le transport grâce à des vésicules spécialisées ( COPI ) entre le réticulum endoplasmique et l'appareil de Golgi, et nombre de ces protéines, telles que p24, une protéine membranaire de type I, ont été décrites comme participant dans des fonctions de régulation. Récemment, des interactions hautement spécifiques protéines-membranes entre p24 et SM C18 impliquées dans la répartition des lipides et l'oligomérisation des protéines ont été décrites. Cette interaction est unique dans le sens où la tête lipidique, autant que la longueur de la chaîne d'acide gras, ont une influence prononcée sur l'affinité entre les lipides et les protéines, et cette haute spécificité suggère un rôle de régulation comparable aux interactions protéines-protéines, ou protéines-acides nucléiques. A notre connaissance, une telle haute spécificité d'interaction lipides-protéines dans les membranes n'a jusqu'à présent pas été observée. L'interaction entre les lipides SM C18 et la protéine p24 implique l'ancrage transmembranaire de la protéine, et cette interaction a été confirmée, même lorsque la protéine est réduite à un domaine transmembranaire de 21 résidus. En particulier, nous projetons de combiner les distances et les angles de torsion qui seront obtenus par des expériences RMN du solide par rotation à l'angle magique ( MAS ) avec des restrictions d'angle dans des échantillons membranaires orientés de manière uniaxiale. La plupart des techniques de RMN du solide que nous allons utiliser n'ont été développées seulement que récemment, et sont continuellement en cours d'amélioration. De plus, la protéine sera étudiée par mutagénèse, spectroscopies optiques, et spectroscopie RMN multidimensionelle en solution dans des micelles, de telle manière qu'un éventail de données structurales, topologiques et fonctionnelles seront obtenues. Lorsque les premiers spectres RMN seront disponibles, les effets remarqués sur l'apparence spectrale ( déplacement chimique, largeur de pic etc... ) de l'interaction spécifique de p24 TM et SM C18 seront étudiés. De plus, les structures lipidiques et protéines-lipides seront définis en termes de paramètres d'ordre et de distances inter-atomiques. Finalement, l'ensemble des informations biochimiques, structurales et de déplacements chimiques permettront de développer un modèle atomique de p24 et du complexe p24- SM C18 dans les bicouches lipidiques.

Coordinateur du projet

Monsieur Burkhad Bechinger (UNIVERSITE DE STRASBOURG) – bechinge@unistra.fr

L'auteur de ce résumé est le coordinateur du projet, qui est responsable du contenu de ce résumé. L'ANR décline par conséquent toute responsabilité quant à son contenu.

Partenaire

CHIMIE - Université de Strasbourg (UdS) UNIVERSITE DE STRASBOURG

Aide de l'ANR 285 000 euros
Début et durée du projet scientifique : - 36 Mois

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