Abstract
The refined structure of a wheat type 2 nonspecific lipid transfer protein (ns-LTP2) liganded with l-alpha-palmitoylphosphatidylglycerol has been determined by NMR. The (15)N-labeled protein was produced in Pichia pastoris. Physicochemical conditions and ligandation were intensively screened to obtain the best NMR spectra quality. This ns-LTP2 is a 67-residue globular protein with a diameter of about 30 A. The structure is composed of five helices forming a right superhelix. The protein presents an inner cavity, which has been measured at 341 A(3). All of the helices display hydrophobic side chains oriented toward the cavity. The phospholipid is found in this cavity. Its fatty acid chain is completely inserted in the protein, the l-alpha-palmitoylphosphatidylglycerol glycerol moiety being located on a positively charged pocket on the surface of the protein. The superhelix structure of the protein is coiled around the fatty acid chain. The overall structure shows similarities with ns-LTP1. Nevertheless, large three-dimensional structural discrepancies are observed for the H3 and H4 alpha-helices, the C-terminal region, and the last turn of the H2 helix. The lipid is orthogonal to the orientation observed in ns-LTP1. The volume of the hydrophobic cavity appears to be in the same range as the one of ns-LTP1, despite the fact that ns-LTP2 is shorter by 24 residues.
Highlights
Plant nonspecific lipid transfer proteins1 were first isolated from spinach leaves and named based on their ability to mediate in vitro the transfer of phospholipids between membranes [1]. ns-LTPs are widely distributed and form a superfamily of related proteins subdivided into two families: the type 1 ns-LTPs and the type 2 ns-LTPs
The biological functions of ns-LTP1 have not yet been clearly determined, the most favored hypothesis being a role in the transport of cutin monomers [4, 5] or in plant defense mechanisms (6 – 8) for ns-LTP1. ns-LTP2 gene expression has been reported in the Zinnia elegans cell differentiation process [9, 10], in barley and rice developing seeds [11, 12], under abiotic stress conditions in barley roots [12], or during nodulation in Vigna unguiculata root hairs [13]
The hydrophobic side chain of Phe-35 is displayed on the cavity surface of the wheat ns-LTP2, whereas in ns-LTP1 the corresponding hydrophilic residue is exposed to the solvent, because of a 180° rotation of the entire helix 3 along its main axis
Summary
Plant nonspecific lipid transfer proteins (ns-LTPs)1 were first isolated from spinach leaves and named based on their ability to mediate in vitro the transfer of phospholipids between membranes [1]. ns-LTPs are widely distributed and form a superfamily of related proteins subdivided into two families: the type 1 ns-LTPs (ns-LTP1) and the type 2 ns-LTPs (nsLTP2) (see Refs. 2 and 3 for review). The refined structure of a wheat type 2 nonspecific lipid transfer protein (ns-LTP2) liganded with L-␣-palmitoylphosphatidylglycerol has been determined by NMR. A set of small monomeric globular proteins was measured under the same conditions to determine the molecular mass calibration.2 Several 31P NMR experiments were performed at 295.2 K on a AMX 400 Bruker spectrometer on a 2 mM sample (pH 3.5, 80 mM NaH2PO4 with 2.5 mM of LPG).
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