STRUCTURE OF A COMPLEX OF THE WHEAT LIPIDE TRANSFERT PROTEIN WITH PHOSPHOLIPIDS

Delphine Charvolin¹, Eva Pebay-Peyroula¹, Claudine Cohen­Addad¹, Didier Marion²

¹Institut de Biologie Structurale, Grenoble, France
²Institut National de Recherche Agronomique, Nantes, France
e-mail: charvolin@godot.ibs.fr

Lipid transfer proteins (LTP) constitute a family of small (around 9 kDa) basic proteins. They show a high degree of homology and contain 8 conserved cysteins, arranged in a Cys­­CysCys­­CysXCys­­Cys­­Cys pattern and forming 4 disulfide bonds. LTP are found abundant in numerous species and various organs of plants: they have been identified in seeds, leaves, stems and shoot apex. Genes encoding for the protein have been located in various tissues as, for example, maize epidermis cell layer, tobacco shoot apex or a barley aleurone layer. There can be several genes per plant genome: thus, four genes were isolated in Brassica Olecea genome. It is also known that LTP are first synthesised as precursory proteins containing 20 to 40 more N-terminal residues, showing that they certainly are secreted to extracellular mediums.

The LTP function is not clearly determined. Initially, the in vitro transfer activity (1) suggested that they could play a role in intracellular lipid transfer. Later, other hypothesis were made: because of their presence in the epidermal cells of the carrot embryo, they were supposed to play a role in the formation of cutin by transferring the hydrophobic cutin monomers (2); it was also suggested that they could be involved in the synthesis of storage lipid during seeds maturation and of cell membranes during embryo's growth (3); the in vitro fungistatic activity (4) and a localisation around cell walls and epidermal cells (5) also suggested that they may play an indirect role in plant's defense mechanisms.

We crystallise wheat LTP in the presence of lyso-myrisroyl-phosphatidyl-choline (LMPC) from 30 % (w/v) PEG 4000 and 0.2 M ammonium acetate in 0.1 M citrate buffer, ph 5.6. Two different crystal forms are obtained in the same drops: P2₁ with cell parameters a= 48.30A, b= 41.90A, c= 51.10A, b= 113.3° or P2₁2₁2₁ with a= 42.29A, b= 53.55A, c= 71.49A, in both cases, two monomers per asymmetric unit. The structure is solved to 2.1 A resolution using X-ray diffraction with an orthorhombic form's crystal. It shows that the protein is linked to two molecules of lipid that cross the protein through its hydrophobic cavity. Previously solved structures of complexed maize (6) or barley (7) LTP showed that they linked to only one molecule of lipid. We will present our results and discuss the consequences of this new information with regard to LTP's function.

1. J.C. Kader, Subcellular Biochemistry (H. J. Hilderson Ed.), Plenum Publishing Corporation (1990) 69-111.
2. P. Sterk, H. Booij, G.A. Schellekens, A. Van Kammen and S.C. De Vries, The Plant Cell 3 (1991) 907-921.
3. L. Sossountzov, L. Ruiz-Avila, F. Vignols, A. Jolliot, V. Arondel, F. Tchang, M. Grosbois, F. Guerbette, E. Miginiac, M. Delseny, P. Puigdomenech and J.C. Kader, The Plant Cell 3 (1991) 923-933.
4. A. Molina, A. Segura and F. Garcia-Olmedo, FEBS 316 (1993) 119-122.
5. J. Pyee, H. Yu and P.E. Kolattukudy, Arch. Biochem. Biophys. 311 (1994) 460-468.
6. D.H. Shin, J.Y. Lee, K.Y. Hwang, K.K. Kim and S.W. Suh, Structure 3 (1995) 189­199.
7. M.H. Lerche, B.B. Kragelund, L.M. Bech and F.M. Poulsen, Structure,5 (1997) 291-306.