STRUCTURE IN SOLUTION OF A 4-HELIX LIPID-BINDING PROTEIN

Because of the low solubility of lipids in water, intercellular and in tracellular pathways of lipid transfer are necessary, e.g., for membra ne formation. The mechanism by which lipids in vivo are transported fr om their site of biogenesis (endoplasmatic reticulum and the chloropla sts) to their place of action is unknown. Several small plant proteins with the ability to mediate transfer of radiolabeled phospholipids in vitro from liposomal donor membranes to mitochondrial and chloroplast acceptor membranes have been isolated, and a protein with this abilit y, the nonspecific lipid transfer protein (nsLTP) isolated from barley seeds (bLTP), has been studied here. The structure and the protein li pid interactions of lipid transfer proteins are relevant for the under standing of their function, and here we present the three-dimensional structure in solution of bLTP as determined by NMR spectroscopy. The H -1 NMR spectrum of the 91-residue protein was assigned for more than 9 7% of the protein H-1 atoms, and the structure was calculated on the b asis of 813 distance restraints from H-1-H-1 nuclear Overhauser effect s, four disulfide bond restraints, from dihedral angle restraints for 66 phi-angles, 61 chi(1) angles, and 2 chi(2) angles, and from 31 sets of hydrogen bond restraints. The solution structure of bLTP consists of four well-defined alpha-helices A-D (A, Cys 3-Gly 19; B, Gly 25-Ala 38; C, Arg 44-Gly 57; D, Leu 63-Cys 73), separated by three short loo ps that are less well defined and concluded by a well defined C-termin al peptide segment with no observable regular secondary structure. For the 17 structures that are used to represent the solution structure o f bLTP, the RMS deviation to an average structure is 0.63 Angstrom +/- 0.04 Angstrom for backbone atoms and 0.93 Angstrom +/- 0.06 Angstrom for all heavy atoms. The secondary structure elements and their locati ons in the sequence resemble those of nsLTP from two other plant speci es, wheat and maize, whose structures were previously determined (Ginc el E et al, 1995, Eur J Biochem 226:413-422; Shin DH et al, 1995, Stru cture 3:189-199). In bLTP, the residues analogous to those in maize ns LTP that constitute the palmitate binding site are forming a similar h ydrophobic cavity and a potential acyl group binding site. Analysis of the solution structure of bLTP and bLTP in complex with a ligand migh t provide information on the conformational changes in the protein upo n ligand binding and subsequently provide information on the mode of l igand uptake and release. In this work, we hope to establish a foundat ion for further work of determining the solution structure of bLTP in complex with palmitoyl coenzyme A, which is a suitable ligand, and sub sequently to outline the mode of ligand binding.