Structural characterization of highly specific membrane protein-lipid interactions involved in trafficking. A novel regulatory mechanism in membrane dynamics? – ProLipIn

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

Contreras FX, Ernst AM, Haberkant P, Björkholm P, Lindahl E, Gönen B, Tischer C, Elofsson A, von Heijne G, Thiele C, Pepperkok R, Wieland F, Brügger B., Molecular recognition of a single sphingolipid species by a proten’s transmembrane domain Nature. 481:525-9 (2012)

2. Ernst, AM, Contreras, FX, Thiele, C, Wieland, F, Brugger, B., Mutual recognition of sphingolipid molecular species in membranes, BBA 1818, 2616-2622

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. To our knowledge such a high specificity of a lipid-protein interaction within membranes has so far not been observed. The interaction between the SM-C18 lipids and the p24 protein involve the transmembrane anchor of the protein and the interaction has been confirmed even when the protein is reduced to a 21-residue transmembrane domain. Here we propose high-resolution structural investigations in bilayer environments using modern solid-state NMR techniques where the structure of the lipid-protein complex will be investigated in membrane environments. In particular we plan to combine the distance and torsion angle information that will be obtained from MAS solid-state NMR experiments with angular restraints from unixially oriented membrane samples. Many of the solid-state NMR techniques that will be used here have only recently been developed and are still in the process of continuous improvement. In addition the protein will be analysed by mutagenesis, optical spectroscopies and multidimensional solution NMR spectroscopy in micellar environments thus a complete data set will be obtained of structural, topological and functional information. Once the first NMR spectra become available the effect on spectral appearance (chemical shifts, line width etc) of the specific interaction of p24 TM with SM C18 will be investigated. Furthermore, the lipid and the lipid-protein structure will be defined in terms of order parameters and interatomic distances. Ultimately, the ensemble of biochemical, structural and chemical shift information will enter a detailed model of p24 as well as of the p24/SM complex in lipid bilayers.