Solution Structure of Plant Nonspecific Lipid Transfer Protein-2 from Rice (Oryza sativa)

Abstract

The three-dimensional structure of rice nonspecific lipid transfer protein (nsLTP2) has been solved for the first time. The structure of nsLTP2 was obtained using 813 distance constraints, 30 hydrogen bond constraints, and 19 dihedral angle constraints. Fifteen of the 50 random simulated annealing structures satisfied all of the constraints and possessed good nonbonded contacts. The novel three-dimensional fold of rice nsLTP2 contains a triangular hydrophobic cavity formed by three prominent helices. The four disulfide bonds required for stabilization of the nsLTP2 structure show a different pattern of cysteine pairing compared with nsLTP1. The C terminus of the protein is very flexible and forms a cap over the hydrophobic cavity. Molecular modeling studies suggested that the hydrophobic cavity could accommodate large molecules with rigid structures, such as sterols. The positively charged residues on the molecular surface of nsLTP2 are structurally similar to other plant defense proteins. Plant nonspecific lipid transfer proteins (nsLTPs)1 have been isolated from a number of plant species including wheat, rice, and barley (1). NsLTPs enhance the intermembrane exchange or transfer of lipid molecules in vitro (2). Biotic and abiotic stresses stimulate nsLTP gene expression (3-5). NsLTPs are known to be involved in the formation of a protective hydrophobic layer over the plant surfaces (5). Despite their ability to help plants to manage stress, the exact mechanism of transport is still unclear. NsLTPs are also involved in other biological activities such as flowering and transportation of cutin and suberin monomers (6). NsLTPs present in cereals play an important role in food chemistry. NsLTPs directly affect dough rheology and breadcrumb texture (6). Reports about the isolation of glycosylated and reduced nsLTP fragments from beer suggest that nsLTPs are involved in froth formation during the malting and brewing processes (7).NsLTPs are divided into two subfamilies, nsLTP1 (molecular mass ϳ9 kDa) and nsLTP2 (molecular mass ϳ7 kDa) (2). NsLTP1 is found primarily in aerial organs, whereas nsLTP2 is expressed in roots. Interestingly, both nsLTP1 and nsLTP2 are found in seeds. NsLTP1 is proposed to transport cutin monomers, whereas nsLTP2 is involved in the transport of the more rigid suberin monomers (6). Three-dimensional structures of nsLTP1 from various sources were determined by x-ray and NMR spectroscopic techniques (8). All nsLTP1s share a common structural fold stabilized by four disulfide bonds. The prominent four helices of nsLTP1 are packed against a flexible C-terminal arm formed by a series of turns. In contrast to many globular proteins, the hydrophobic side chains of nsLTP1 do not form a rigid hydrophobic core but instead form a hydrophobic cavity at the interior of the protein. Recently, we have purified nsLTP2 from rice. The amino acid sequence, disulfide bond pattern and stability have been determined (TrEMBL ID P83210) (9). Rice nsLTP2 contains 69 residues and has less than 30% sequence identity wi...

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

“…The Tm of ns-LTP1 is about 100°C 187,234,235,259 , while an even higher temperature is required for denaturation of ns-LTP2, as well as higher concentrations of denaturants 296 . Differences in the hydrophobic cavity shape and flexibility explain why ns-LTP2 has the ability to bind not only linear molecules such as ns-LTP1, but planar sterols as well, and why it possesses higher lipid transfer activity 75,296 . The level of wheat seed ns-LTP2 transfer activity is at least five times higher than that of wheat ns-LTP1s 75 .…”

A Review: The Role of Barley Seed Pathogenesis-Related Proteins (PRs) in Beer Production

“…The protein ns-LTP2 has not been as studied as ns-LTP1. However, there is increasing literature on cereal seed ns-LTP2 structure, stability and lipid-binding ability in the last few years 20,32,85,99,110,138 to elucidate its possible technological function in cereal processing. In contrast to ns-LTP1, which is expressed in the aleurone layer late in grain development and in the early stages of germination, ns-LTP2 is expressed during the early stages of grain development.…”

Functionality of lipids and lipid-protein interactions in cereal-derived food products

“…Both LTP1s and LTP2s are alpha-helix proteins [18,22]. In LTP1, the four helices form a bundle-like shape surrounded in part by a C-terminal polypeptide composed by turns forming a saxophone-like structure ( figure 6).…”

“…The hydrophobic cavity is a triangular hollow box and not a tunnel. A part of LTP2 and LTP1 structures can be superimposed [22]. The tunnel of LTP1s is highly flexible and its volume increases[MG1] to bind one or two molecules of monoacylated lipids or a diacyl phospholipid [18] ( figure 7).…”

“…An hydrophobic tunnel, forming the lipid binding site (see below) follows the long axis of the protein. The rice LTP2 is the only protein of this family for which a structure has been recently determined [22]. LTP2 displays three main helices and two single turn helices ( figure 6).…”