Structure in solution of a four‐helix lipid binding protein

Abstract

Because of the low solubility of lipids in water, intercellular and intracellular pathways of lipid transfer are necessary, e.g., for membrane formation. The mechanism by which lipids in vivo are transported from their site of biogenesis (endoplasmatic reticulum and the chloroplasts) 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 ability, the nonspecific lipid transfer protein (nsLTP) isolated from barley seeds (bLTP), has been studied here. The structure and the protein lipid interactions of lipid transfer proteins are relevant for the understanding of their function, and here we present the three-dimensional structure in solution of bLTP as determined by NMR spectroscopy. The ' H NMR spectrum of the 91-residue protein was assigned for more than 97% of the protein ' H atoms, and the structure was calculated on the basis of 813 distance restraints from 'H-'H nuclear Overhauser effects, four disulfide bond restraints, from dihedral angle restraints for 66 @-angles, 61 x' angles, and 2 x ' angles, and from 31 sets of hydrogen bond restraints. The solution structure of bLTP consists of four well-defined a-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 loops that are less well defined and concluded by a well defined C-terminal peptide segment with no observable regular secondary structure. For the 17 structures that are used to represent the solution structure of bLTP, the RMS deviation to an average structure is 0.63 A f 0.04 A for backbone atoms and 0.93 A k 0.06 A for all heavy atoms. The secondary structure elements and their locations in the sequence resemble those of nsLTP from two other plant species, wheat and maize, whose structures were previously determined (Gincel E et al, 1995, Eur JBiochern 226:413-422; Shin DH et al, 1995, Structure3: 189-199). In bLTP, the residues analogous to those in maize nsLTP that constitute the palmitate binding site are forming a similar hydrophobic cavity and a potential acyl group binding site. Analysis of the solution structure of bLTP and bLTP in complex with a ligand might provide information on the conformational changes in the protein upon ligand binding and subsequently provide information on the mode of ligand uptake and release. In this work, we hope to establish a foundation for further work of determining the solution structure of bLTP in complex with palmitoyl coenzyme A, which is a suitable ligand, and subsequently to outline the mode of ligand binding.

1H NMR and fluorescence studies of the complexation of DMPG by wheat non‐specific lipid transfer protein. Global fold of the complex

“…E-mail : sodano@cnrs-orleans.fr helices and a C-terminal fragment organized around a long internal cavity which has been suggested to be the lipid binding site. This type of organization was then confirmed with the maize ns-LTP structure that we established in solution [3] and in crystal by Shin et al [4] and more recently with the barley ns-LTP structure [5]. The crystal structure of the complex of the maize protein with palmitic acid [4], the solution structure of its complex with l-palmitoyl-2-lysophosphatidylcholine [3] and the barley ns-LTP complex with palmitoylCoA [6] demonstrated the capacity of ns-LTPs to incorporate a long fatty acid chain in their internal cavity.…”

“…The C-terminal region was devoid of any regular hydrogen bonded canonical structure except for a ß-turn involving residues 82-85 and ran parallel to H4 from residues 77 to 83 and then to H3 from residue 84 to the C-terminal residue. The protein scaffold which was common to all available ns-LTP structures [3][4][5] was maintained by a network of four disulfide bridges distributed in pairs on two protein poles, providing great stability to this protein. The RMSD calculated on the heavy atoms of the backbone of the four helices relative to the mean structure is 0.75 A which is indicative of a moderate resolution solution structure.…”

A Review: Biological and Technological Functions of Barley Seed Pathogenesis-Related Proteins (PRs)

“…The ns-LTPs are all helical proteins, stabilized by four disulfide bridges 75,120,193 . The ns-LTP1 structural fold is characterized by four α-helices separated by short turns and held together with four disulphide bridges, and a flexible nonstructured C-terminal arm 120 (Fig. 13).…”

Barley Grain Non-specific Lipid-Transfer Proteins (ns-LTPs) in Beer Production and Quality

“…The three-dimensional structures of ns-LTP1 from barley 68 , wheat 53 , rice 86 and maize grain 136 have been resolved. They all exhibit a similar fold, differing only in details 121 .…”